Fabric and home care products

ABSTRACT

This invention relates to fabric and home care products comprising one or more cold water proteases and processes for making and using such products. Such compositions provide improved cleaning and freshness. Such cold water proteases may be derived from parent enzymes, including BPN′ subtilisin and subtilisin derived from  Bacillus lentus,  by substitution, insertion and/or deletion of one or more of the parent enzymes&#39; amino acids.

FIELD OF INVENTION

This invention relates to fabric and home care products comprising cold water proteases as well as methods of making and using such fabric and home care products.

BACKGROUND OF THE INVENTION

Detergent manufacturers incorporate proteases into their products to provide good cleaning of stains (such as blood). However, given the sustainability and consumer trends to lower wash temperatures it is proving increasingly difficult to deliver consumer acceptable benefits and there remains a need to improve the cleaning and freshness profile of these laundry detergent compositions. The Inventors have found that additionally incorporating certain cold water proteases into a fabric and home care products, for example, a laundry detergent composition that comprises a hueing agent, a cleaning polymer and/or a perfume capsule, improves the whiteness, whiteness perception, and/or freshness.

SUMMARY OF THE INVENTION

This invention relates to fabric and home care products comprising one or more cold water proteases and processes for making and using such products. Such compositions provide improved cleaning and freshness. Such cold water proteases may be derived from parent enzymes, including BPN′ subtilisin and subtilisin derived from Bacillus lentus, by substitution, insertion and/or deletion of one or more of the parent enzymes' amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a plasmid map of pHPLT-BPN′-v3.

FIG. 2 provides a plasmid map of pHPLT-BPN′-v3+S78N.

FIG. 3 provides a plasmid map of pHPLT-BPN′ partial opt.

FIG. 4 provides a plasmid map of pHPLT-BPN′-v36.

FIG. 5 provides an alignment of the mature reference subtilisin proteases including: BPN′ (SEQ ID NO:2) and GG36 (SEQ ID NO:755). Each amino acid position of each protease variant described herein, including each cold water protease variant, is numbered according to the numbering of the corresponding amino acid position in the amino acid sequence of Bacillus amyloliquefaciens subtilisin protease BPN′ (SEQ ID NO:2), as shown in FIG. 5, as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin protease BPN′ amino acid sequence. Thus, unless otherwise specified herein, substitution positions are given in relationship to BPN′.

FIG. 6 provides map of pHPLT-GG36.

FIG. 7 provides a map of pRA68.

FIG. 8 provides a map of pRA96.

FIG. 9 provides a map of pAC-FNAre.

FIG. 10 is a schematic representation of method or targeted ISD library construction.

FIG. 11 provides a map of pAC-FNA10.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein “fabric and home products” means products or devices generally intended to be used or consumed in the form in which they are sold and that are for treating fabrics, hard surfaces and any other surfaces in the area of fabric and home care, including: air care including air fresheners and scent delivery systems, car care, dishwashing, fabric conditioning (including softening and/or freshening), laundry detergency, laundry and rinse additive and/or care, hard surface cleaning and/or treatment including floor and toilet bowl cleaners, and other cleaning for consumer or institutional use.

As used herein, the term “cleaning and/or treatment composition” is a subset of fabric and home care products that includes, unless otherwise indicated fabric & home care products. Such products include, but are not limited to, products for treating fabrics, hard surfaces and any other surfaces in the area of fabric and home care, including: air care including air fresheners and scent delivery systems, car care, dishwashing, fabric conditioning (including softening and/or freshening), laundry detergency, laundry and rinse additive and/or care, hard surface cleaning and/or treatment including floor and toilet bowl cleaners, granular or powder-form all-purpose or “heavy-duty” washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use: car or carpet shampoos, bathroom cleaners including toilet bowl cleaners; as well as cleaning auxiliaries such as bleach additives and “stain-stick” or pre-treat types, substrate-laden products such as dryer added sheets.

As used herein, the term “fabric and/or hard surface cleaning and/or treatment composition” is a subset of cleaning and treatment compositions that includes, unless otherwise indicated, granular or powder-form all-purpose or “heavy-duty” washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, car or carpet shampoos, bathroom cleaners including toilet bowl cleaners; fabric conditioning products including softening and/or freshening that may be in liquid, solid and/or dryer sheet form; as well as cleaning auxiliaries such as bleach additives and “stain-stick” or pre-treat types, substrate-laden products such as dryer added sheets. All of such products which are applicable may be in standard, concentrated or even highly concentrated form even to the extent that such products may in certain aspect be non-aqueous.

As used herein, articles such as “a” and “an” when used in a claim, are understood to mean one or more of what is claimed or described.

As used herein, the terms “include”, “includes” and “including” are meant to be non-limiting.

As used herein, the term “solid” includes granular, powder, bar and tablet product forms.

As used herein, the term “fluid” includes liquid, gel, paste and gas product forms.

As used herein, the term “situs” includes fabrics, garments, and/or hard surfaces.

Unless otherwise noted, all component or composition levels are in reference to the active portion of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources of such components or compositions.

All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated.

It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

Fabric and Home Products Comprising One or More Proteases

A composition comprising a protease, said protease being selected from the group consisting of:

-   a) a cold water protease having a performance index greater than 1,     at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least     1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at     least 2 from 1 to about 10, from 1 to about 8 or even from 1 to     about 5 on BMI at pH 8 and 60° F. when compared to an enzyme having     SEQ ID NO:4, as defined in Test Method 2 and/or Test Method 3; -   b) a cold water protease having a performance index at least 1, at     least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5,     at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2     from 1 to about 10, from 1 to about 8 or even from 1 to about 5 on     BMI at pH 8 and 60° F. when compared to an enzyme having SEQ ID     NO:6, as defined in Test Method 2 and/or Test Method 3; -   c) a cold water protease, said cold water protease being selected     from the group consisting of:     -   (i) a variant of a parent protease, said parent protease having         at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%         or 99% or even 100% identity to SEQ ID NO:4, said variant         comprising a group of mutations selected from the following         groups of mutations: P210S, P210S-N218A,         S063T-S078N-S101A-S183T-T244N, N061A-S078N-S224A,         S053G-S078N-P129T-Q185T, S063T-S078N-S101A, S078N-P129T,         S063T-S078N-S101A-S183T and S063T-S078N-S101A-T2441;     -   (ii) a variant of a parent protease, said parent protease having         at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%         or 99% or even 100% identity to SEQ ID NO:2, said variant         comprising a group of mutations selected from the following         groups of mutations: G097A-I111V-M124V-Y217Q,         G097A-I111V-Y167A-Y217Q,         S024G-N025G-N061P-G097A-S101N-G128S-Y217Q,         S024G-N025G-S053G-N061P-G097A-S101N-G128A-V203Y-Y217Q,         S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128S-V203Y-Y217Q,         V068A-A092G-Y217Q, N061P-G097A-S101N-G128A-P201S-Y217Q,         S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q,         S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q,         S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q,         S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,         N025G-G097A-S101N-G128A-Y217Q,         N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,         N025G-S053G-N061P-S078N-G128A-Y217Q,         N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,         N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,         N025G-S078N-G097A-S101N-G128A-Y217Q,         N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         N025G-T055P-N061P-S078N-S101N-G128A-Y217Q,         N061P-S101N-G128A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q,         S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,         S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,         S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,         S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,         S024G-N025G-T055P-G097A-G128A-Y217Q,         S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-S053G-N061P-G097A-G128A-Y217Q,         S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,         S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,         S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,         S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,         S024G-T055P-N061P-G097A-G128A-Y217Q,         S053G-G097A-S101N-G128A-Y217Q,         S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,         S053G-N061P-S078N-G097A-G128A-Y217Q,         S053G-S078N-G097A-S101N-G128A-Y217Q,         S053G-T055P-G097A-S101N-G128A-Y217Q,         S053G-T055P-N061P-S101N-G128A-Y217Q,         S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,         T055P-G097A-S101N-G128A-Y217Q,         T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,         S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,         T055P-N061P-S078N-G128A-Y217Q and         S24G-S53G-S78N-S101N-G128A-Y217Q;     -   (iii) a variant of a parent protease, said parent having at         least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or         99% or even 100% identity to SEQ ID NO:2, said variant         comprising three, four, five or six of the following mutations         X24G/R, X53G, X78N, X101N, X128A/S and X217L/Q; said variant         optionally comprising at least one group of mutations selected         from the following groups of mutations: A116V, G160S, I111L,         I115V, N109S, N117M, P005G, Q059V, T164S, Y262M, A015Q, A015S,         A098E, A098N, A098S, A098T, A098V, A098Y, A114S, A114T, A116G,         A116L, A116S, A116T, A116W, A133G, A133H, A133T, A133V, A137G,         A137I, A137L, A137S, A137T, A138S, A216E, A216F, A216V, D099S,         D181E, F261A, F261Q, G024F, G024I, G024Q, G024Y, G097S, G160T,         G211L, G211V, H017F, H017W, H039V, H226A, I031V, I111V, I268V,         K170R, K265R, L016Q, L016T, L135M, L209T, L209V, L233M, L257T,         L257V, L267A, L267V, N025A, N025I, N025Q, N025R, N025T, N025V,         N101I, N101Q, N101S, N109A, N109G, N109H, N109L, N109M, N109Q,         N109T, N117Q, N184A, N184L, N184T, N184W, N212G, N212L, N212V,         N243P, N252G, N252M, P005T, P014S, P040G, P040L, P040Q, P129A,         P129S, P172G, P172S, P194Q, P210A, P210S, Q185F, Q185G, Q185I,         Q185M, Q185N, Q185S, Q275H, R186K, S009A, S009G, S009H, S009M,         S018T, S130T, S132N, S145K, S159T, S161I, S161K, S161N, S161T,         S162I, S162M, S162Y, S163G, S182F, S182G, S182V, S182W, S183F,         S183L, S183M, S183T, S183V, S183W, S224A, S236T, S249V, T022A,         T022G, T022Q, T022V, T208V, T242S, T253N, T253S, T254A, T254S,         T255L, T255S, T255V, V004A, V004P, V004W, V084C, V139C, V165M,         V203F, Y021K, Y021N, Y021T, Y021V, Y167F, Y171F, Y214F, Y262F,         Y262T, A088T-L257G, A116T-A128S, N061S-N109G-A128S-N243V-S260P,         S009T-N109G-A128S-K141R-N243V,         S009T-S018T-Y021N-N109G-A128S-K141R, S162G-K256R, A088T-N243V,         G024E-A116T, K043Y, N076D-A116T, N218S-S248N, S033T-N243V,         S033T-S063G, S248N-L257G, A001E-S249A, A088T-A116T, A088T-A128S,         A088T-G131H, A088T-N109G, A088T-S248N, A088T-S249A, A116T-N243V,         A116T-T158S, A128S, A128S-K256R, A128S-L257G, A128S-N243V,         A128S-S248N, A128S-T158S, G024E-A088T, G024E-A128S, G024E-G131H,         G024E-K256R, G024E-L257G, G024E-N218S, G024E-N243V, G024E-S162G,         G024E-S249A, G024E-T158S, G131H, G131H-K256R, G131H-S249A,         K043Y-A088T, K043Y-A116T, K256R, N076D-K256R, N109G,         N109G-A116T, N109G-A128S, N109G-A128S-N243V-K256R,         N109G-A128S-N243V-S248A, N109G-G131H, N109G-K256R, N109G-L257G,         N109G-N218S, N109G-N243V, N109G-S248N, N218S-L257G, N243V,         N243V-K256R, N243V-L257G, N243V-S248N, N243V-S249A, Q103H-A128S,         Q103H-G131H, Q103H-K256R, Q103H-L257G, Q103H-N243V, Q103H-S248N,         Q103H-S249A, Q103H-T158S, Q206D-N243V, S033T-A128S, S033T-K256R,         S033T-N076D, S033T-N218S, S033T-S248N, S033T-T158S, S063G-A128S,         S063G-K256R, S063G-N243V, S063G-S162G, S063G-T158S, S248N-K256R,         S249A, T158S-N243V, T158S-S249A, A088T-A116T-N243V-K256R-L257G,         A088T-A116T-N243V-L257G, A088T-T158S-N218S-K256R,         A088T-T158S-N218S-N243V-L257G,         A088T-A116T-T158S-N218S-N243V-K256R-L257G,         A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G,         A088T-N109G-A116T-T158S-S248N-K256R-L257G,         A088T-N109G-T158S-L257G,         A114S-A116T-N218S-N243V-S248N-K256R-L257G, A116T-T158S-K256R,         A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-T158S,         A088T-N109G-A116T-G131H-L257G,         A088T-N109G-A116T-T158S-N243V-S248N-L257G,         A088T-N109G-N243V-L257G, A088T-N109G-N243V-S248N,         A088T-N109G-T158S-N243V-L257G,         A088T-N109G-T158S-N243V-S248N-L257G, A116T-T158S-S248N-L257G,         Y006H-A116T-G131H-S248N, A088T-A116T-G131H-T158S-N218S-N243V,         A088T-A116T-G131H-T158S-N243V,         A088T-A116T-G131H-T158S-N243V-K256R-L257G,         A088T-A116T-N218S-N243V-K256R-L257G,         A088T-A116T-S248N-K256R-L257G, A088T-A116T-T158S-N218S-N243V,         A088T-A116T-T158S-N243V-K256R-L257G,         A088T-A116T-T158S-N243V-S248N-L257G,         A088T-G131H-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-T158S-L257G,         A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G,         A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,         A088T-N109G-A116T-T158S-S248N-L257G,         A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G,         A088T-N109G-K256R, A088T-N109G-N243V-S248N-L257G,         A088T-N109G-T158S-K256R A088T-N109G-T158S-N243V,         A088T-T158S-N243V-K256R-L257G, A116T-N218S-N243V-L257G-N269S,         A116T-T158S-K256R-L257G, N109G-A116T-K256R-L257G,         N109G-A116T-N243V, N109G-A116T-T158S-N243V-K256R-L257G,         N109G-G131H-L257G, N109G-G131H-S248N-K256R-L257G,         N109G-G131H-T158S-K256R-L257G, S003P-A116T-T158S-S248N-K256R,         T158S-S248N-K256R, A088T-A116T-G131H-N243V-K256R,         A088T-A116T-G131H-S248N-K256R-L257G,         A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G,         A088T-A116T-S248N-L257G, A088T-A116T-T158S-N218S,         A088T-A116T-T158S-N218S-K256R-L257G,         A088T-A116T-T158S-N218S-L257G, A088T-G131H-N243V-L257G,         A088T-G131H-T158S-S248N-L257G, A088T-N109G-A116T,         A088T-N109G-A116T-G131H-N218S,         A088T-N109G-A116T-G131H-N218S-S248N-L257G,         A088T-N109G-A116T-G131H-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,         A088T-N109G-A116T-N218S-N243V-K256R,         A088T-N109G-A116T-N218S-N243V-L257G,         A088T-N109G-A116T-N243V-S248N-K256R,         A088T-N109G-A116T-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-T158S-N243V-L257G,         A088T-N109G-G131H-T158S-N243V-S248N-K256R,         A088T-N109G-G131H-T158S-W241R-S248N-K256R,         A088T-N109G-K256R-L257G, A088T-N109G-L257G, A088T-N109G-N243V,         A088T-N109G-N243V-K256R, A088T-N109G-N243V-K256R-L257G,         A088T-N109G-S248N-K256R, A088T-N109G-T158S-N218S-K256R-L257G,         A088T-N109G-T158S-N218S-N243V-S248N-K256R,         A088T-N109G-T158S-N243V-K256R,         A088T-N109G-T158S-N243V-K256R-L257G,         A088T-N109G-T158S-N243V-S248N-A274D,         A088T-N109G-T158S-S248N-L257G, A088T-T158S-K256R,         A088T-T158S-N218S-N243V-K256R-L257G, A088T-T158S-N243V-L257G,         A116T-G131H-N218S-N243V-S248N, A116T-G131H-S248N-L257G,         A116T-S248N-K256R-L257G, A116T-T158S-N218S-N243V-K256R,         A116T-T158S-N218S-S248N-L257G-Q271R,         A116T-T158S-N243V-K256R-L257G, A116T-T158S-N243V-S248N-L257G,         G131H-S248N, G131H-T158S-I234T-N243V-K256R,         G131H-W241L-N243V-S248N-K256R,         N109G-A116T-G131H-A137V-T158S-S248N-K256R-L257G,         N109G-A116T-G131H-A151S-N218S-K256R-L257G,         N109G-A116T-G131H-T158S-N218S-N243V-K256R,         N109G-A116T-G131H-T158S-N218S-S248N,         N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-S248N,         N109G-A116T-T158S-L257G, N109G-A116T-T158S-N218S-W241R-N243V,         N109G-A116T-T158S-N243V-S248N-L257G,         N109G-A116T-T158S-S248N-K256R-L257G,         N109G-A116T-T158S-S248N-L257G, N109G-G131H-N218S-L257G,         N109G-G131H-N218S-S248N-K256R-L257G,         N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T,         N109G-N243V-L257G, N109G-T158S-N218S-K256R-L257G,         N109G-T158S-N218S-L257G, N109G-T158S-S248N-K256R,         P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E,         S003F-A088T-N109G-A116T-T158S-N243V-K256R-L257G,         V004A-A088T-A116T-T158S-N218S,         V004A-N109G-A116T-G131H-S248N-K256R-L257G,         V004L-A116T-N218S-N243V-S248N-L257G,         Y006H-N109G-N218S-N243V-S248N, A001T-A116T-T158S-N243V-L257G,         A088T-A116T-G131H-L257G, A088T-A116T-G131H-N218S-L257G,         A088T-A116T-G131H-N218S-S248N-K256R-L257G,         A088T-A116T-G131H-N218S-S248N-L257G,         A088T-A116T-G131H-N243V-K256R-L257G,         A088T-A116T-G131H-N243V-L257G, A088T-A116T-G131H-N243V-S248N,         A088T-A116T-G131H-T158S-K256R-L257G,         A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S,         A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T,         A088T-A116T-G131H-T158S-N218S-S248N-K256R,         A088T-A116T-G131H-T158S-N218S-S248N-L257G,         A088T-A116T-G131H-T158S-N243V-S248N-K256R,         A088T-A116T-G131H-T158S-S248N, A088T-A116T-K256R,         A088T-A116T-K256R-L257G, A088T-A116T-N218S-N243V-L257G,         A088T-A116T-N243V-K256R, A088T-A116T-N243V-S248N-K256R-L257G,         A088T-A116T-S248N-K256R, A088T-A116T-T158S-K256R,         A088T-A116T-T158S-N218S-N243V-K256R,         A088T-A116T-T158S-N218S-N243V-K256R-N269S,         A088T-A116T-T158S-N218S-N243V-S248N,         A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-A116T-T158S-N243V-K256R, A088T-A116T-T158S-N243V-L257G,         A088T-A116T-T158S-N243V-S248N-K256R,         A088T-A116T-T158S-N243V-S248N-K256R-L257G,         A088T-A116T-T158S-S248N-K256R,         A088T-A116T-V143A-N218S-S248N-K256R,         A088T-A116T-V147I-T158S-N218S-N243V-L257G,         A088T-G131H-K256R-L257G, A088T-G131H-N218S-N243V-S248N,         A088T-G131H-N218S-S248N-L257G, A088T-G131H-S248N-K256R-L257G,         A088T-G131H-T158S-L257G, A088T-G131H-T158S-N218S-K256R,         A088T-G131H-T158S-N218S-N243V-K256R-L257G,         A088T-G131H-T158S-N218S-N243V-L257G,         A088T-G131H-T158S-N218S-S248N, A088T-G131H-T158S-N243V,         A088T-G131H-T158S-N243V-S248N,         A088T-G131H-T158S-N243V-S248N-K256R,         A088T-G131H-T158S-N243V-S248N-L257G,         A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,         A088T-N109G-A116T-G131H-N218S-L257G,         A088T-N109G-A116T-G131H-N218S-N243V,         A088T-N109G-A116T-G131H-N218S-N243V-K256R-L257G,         A088T-N109G-A116T-G131H-N218S-N243V-L257G,         A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-N243V,         A088T-N109G-A116T-G131H-N243V-L257G,         A088T-N109G-A116T-G131H-N243V-S248N-L257G,         A088T-N109G-A116T-G131H-S248N,         A088T-N109G-A116T-G131H-S248N-K256R,         A088T-N109G-A116T-G131H-S248N-L257G,         A088T-N109G-A116T-G131H-T158S-L257G,         A088T-N109G-A116T-G131H-T158S-N218S,         A088T-N109G-A116T-G131H-T158S-N218S-S248N-K256R,         A088T-N109G-A116T-G131H-T158S-N218T-N243V,         A088T-N109G-A116T-G131H-T158S-N243V-K256R,         A088T-N109G-A116T-G131H-T158S-N243V-K256R-L257G,         A088T-N109G-A116T-G131H-T158S-N243V-S248N,         A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R,         A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-T158S-S248N-L257G,         A088T-N109G-A116T-N218S, A088T-N109G-A116T-N218S-L257G,         A088T-N109G-A116T-N218S-N243V,         A088T-N109G-A116T-N218S-N243V-S248N-L257G,         A088T-N109G-A116T-N218S-S248N-K256R,         A088T-N109G-A116T-N218T-K256R,         A088T-N109G-A116T-N218T-K256R-L257G, A088T-N109G-A116T-N243V,         A088T-N109G-A116T-N243V-K256R-L257G,         A088T-N109G-A116T-N243V-K256R-L257G-N269D,         A088T-N109G-A116T-S248N-K256R, A088T-N109G-A116T-T158S,         A088T-N109G-A116T-T158S-N218S-L257G,         A088T-N109G-A116T-T158S-N218S-N243V,         A088T-N109G-A116T-T158S-N218S-N243V-K256R,         A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G,         A088T-N109G-A116T-T158S-N218S-N243V-L257G,         A088T-N109G-A116T-T158S-N218S-S248N,         A088T-N109G-A116T-T158S-N243V,         A088T-N109G-A116T-T158S-N243V-K256R,         A088T-N109G-A116T-T158S-N243V-K256R-L257G,         A088T-N109G-G131H-L257G, A088T-N109G-G131H-N218S-K256R-L257G,         A088T-N109G-G131H-N218S-N243V-K256R,         A088T-N109G-G131H-N218S-N243V-L257G,         A088T-N109G-G131H-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-G131H-N243V, A088T-N109G-G131H-N243V-L257G,         A088T-N109G-G131H-N243V-S248N-K256R,         A088T-N109G-G131H-N243V-S248N-L257G,         A088T-N109G-G131H-S248N-L257G, A088T-N109G-G131H-T158S-L257G,         A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R,         A088T-N109G-G131H-T158S-N243V,         A088T-N109G-G131H-T158S-N243V-K256R,         A088T-N109G-G131H-T158S-N243V-K256R-L257G,         A088T-N109G-G131H-T158S-N243V-L257G, A088T-N109G-N218S-K256R,         A088T-N109G-N218S-N243V-S248N-L257G,         A088T-N109G-N218S-S248N-K256R-L257G,         A088T-N109G-N243V-S248N-K256R-L257G,         A088T-N109G-N243V-S248N-L257G-I268V,         A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S-N218S-K256R,         A088T-N109G-T158S-N218S-N243V-L257G,         A088T-N109G-T158S-N243V-K256R-I268V,         A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N218S-N243V,         A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-S248N,         A088T-N218S-S248N-L257G, A088T-N243V-K256R, A088T-N243V-L257G,         A088T-S145T-T158S-S248N, A088T-T158S-L257G,         A088T-T158S-N218S-S248N-L257G,         A088T-T158S-N243V-K256R-L257G-Q271H, A088T-T158S-S248N,         A088T-V143A-T158S-K256R, A116T-G131H-K256R, A116T-G131H-N218S,         A116T-G131H-N243V, A116T-G131H-N243V-K256R,         A116T-G131H-N243V-L257G, A116T-G131H-S248N-K256R,         A116T-G131H-T158S-N218S-I234T-N243V-S248N-K256R,         A116T-G131H-T158S-N243V-L257G,         A116T-G131H-T158S-N243V-S248N-K256R,         A116T-G131H-V143F-T158S-N218S, A116T-L257G, A116T-N218S,         A116T-N218S-L257G, A116T-N218S-N243V-L257G, A116T-N243V-K256R,         A116T-N243V-S248N, A116T-N243V-S248N-K256R-L257G, A116T-S248N,         A116T-T158S-N218S-N243V, A116T-T158S-N218S-S248N,         A116T-T158S-N243V, A116T-T158S-N243V-K256R,         A116T-T158S-N243V-L257G, A116T-T158S-N243V-S248N,         A116T-T158S-S248N-K256R-L257G,         A116T-V149I-T158S-N243V-S248N-K256R-Q271H,         G131H-N218S-N243V-L257G, G131H-N243V, G131H-N243V-S248N-K256R,         G131H-T158S, G131H-T158S-N218S-N243V-K256R,         G131H-T158S-N243V-K256R-L257G, G131H-T158S-N243V-S248N-L257G,         N109G-A116T-G131H-N218S-K256R-L257G,         N109G-A116T-G131H-N218S-L257G,         N109G-A116T-G131H-N218S-N243V-K256R-L257G,         N109G-A116T-G131H-N218S-S248N-K256R,         N109G-A116T-G131H-N243V-K256R, N109G-A116T-G131H-N243V-L257G,         N109G-A116T-G131H-N243V-S248N-K256R-L257G,         N109G-A116T-G131H-S248N, N109G-A116T-G131H-S248N-I268V,         N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,         N109G-A116T-G131H-T158S-N218S-S248N-L257G,         N109G-A116T-G131H-T158S-S248N,         N109G-A116T-G131H-T158S-S248N-K256R, N109G-A116T-N218S,         N109G-A116T-N218S-N243V-K256R,         N109G-A116T-N218S-N243V-K256R-L257G,         N109G-A116T-N218S-S248N-L257G, N109G-A116T-N243V-K256R,         N109G-A116T-N243V-S248N-K256R-L257G, N109G-A116T-S248N-L257G,         N109G-A116T-T158S-G211V-N243V-S248N-K256R,         N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S,         N109G-A116T-T158S-N218S-N243V-K256R-L257G,         N109G-A116T-T158S-N218S-N243V-L257G,         N109G-A116T-T158S-N218S-N243V-S248N-L257G,         N109G-A116T-T158S-N218S-S248N-K256R-L257G,         N109G-A116T-T158S-N243V, N109G-A116T-T158S-Q275R,         N109G-G131H-A137V-T158S-N218S-S248N, N109G-G131H-N218S-K237N,         N109G-G131H-N218S-N243V-K256R-L257G,         N109G-G131H-N218S-S248N-K256R, N109G-G131H-N243V-K256R-L257G,         N109G-G131H-S145F-N218S-N243V-K256R-L257G,         N109G-G131H-S248N-K256R, N109G-G131H-S248N-L257G,         N109G-G131H-T158S-K256R, N109G-G131H-T158S-N218S-N243V-K256R,         N109G-G131H-T158S-N243V, N109G-G131H-T158S-N243V-K256R-L257G,         N109G-G131H-T158S-N243V-L257G, N109G-G131H-T158S-S248N-L257G,         N109G-G131H-T158S-S248N-Q271R, N109G-N218S-L257G,         N109G-N218S-N243V, N109G-N243V-K256R-L257G,         N109G-N243V-S248N-K256R-L257G, N109G-T158S-I268V,         N109G-T158S-K256R, N109G-T158S-N218S-N243V-K256R-L257G,         N109G-T158S-N218S-S248N-L257G, N109G-T158S-N243V,         N109G-T158S-N243V-K256R-L257G, N109G-T158S-N243V-S248N,         N109S-A116T-S248N, N218S, N218S-N243V-S248N-K256R-L257G,         N218S-S248N-L257G, N243V-K256R, N243V-S248N-K256R,         N243V-S248N-K256R-L257G,         S105P-A116T-T158S-N218S-N243V-S248N-K256R, S248N,         T158S-N243V-K256R, T158S-N243V-L257G, S018F-S162L, S018P-D120N,         P014T-S037T, S009T-K141R, S161P-S162L,         N61P-S63G-N109Q-A128S-S224A-N243V,         A88T-N109G-A114S-A116T-A128S-N243V,         A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V,         N109G-A128S-S183V, N109M-A128S-S224A,         A88T-N109S-A116T-A128S-S224A-N243V, N109Q-A128S-S224A-N243V,         A88T-N109M-A116T-A128S-S224A-N243V, S63G-A128S,         N109S-A128S-S224A-N243V, A88T-N109G-A116T-N243V,         N61S-N109G-N243V, N101Q-N109Q-A128S-S224A-N243V,         A88T-N109G-A116T-T158S-N243V-K256R, N109G-A116T, A88T-N109G,         N61G-N109G-N243V, N109G-A128S-P129S-S130T-S224A-N243V,         A88T-N109Q-A116T-A128S-S224A-N243V, N062L-S063N-Q217L,         N062S-S063R-Q217E, N062S-S063L-Q217L, S063G, S063G-Q217L, S063N         and S063N-Q217L; -   d) a cold water protease, said cold water protease being selected     from the group consisting of:     -   (i) a variant of a parent protease, said parent protease having         at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%         or 99% or even 100% identity to SEQ ID NO:4, said variant         comprising a group of mutations selected from the following         groups of mutations: E156S-P210S, P210S-N218S,         Y104F-E156S-P210I, E156A-P210S-N218S, N218A,         N061A-S078N-S087E-S224A, Q059V-S078N-G211A, Q059V-S078N-V147Q,         Q059V-S078N, Q059V-S078N-I108V-N252Q, S053G-S078N-P129T,         S078N-G211A, S078N-Q185T, S078N-V147Q, S078N, S078N-S224A,         S078N-S224A-A274D, S078N-I108V-V147Q, N061A-S078N-S224A,         S053G-S078N-P129T-Q185T and S078N-P129T;     -   (ii) a variant of a parent protease, said parent protease having         at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%         or 99% or even 100% identity to SEQ ID NO:2, said variant         comprising a group of mutations selected from the following         groups of mutations: G097A-I111V-Y217Q, I111V-G128A-Y217Q,         I111V-M124V-Y167A, I111V-M124V-Y217Q, L096T-G097A-Y217Q,         N062Q-G097A-I111V, S053G-N061P-G097A-S101N-G128S-V203Y-Y217Q,         S089Y-M124V-Y217Q, V068A-I111V-Y217Q, G097A-I111V-M124V,         G097A-L126A-Y217Q, G097A-M124V-Y217Q, I111V-Y167A-Y217Q,         M124V-Y167A-Y217Q, P052L-V068A-G097A, S089Y-I111V-M124V,         V068A-A092G-G097A, V068A-A092G-I111V, V068A-G097A-I111V,         V068A-S089Y-I111V, Y104N-G128A-Y217Q, G097A-G128A-P210S-Y217Q,         G097A-G128S-P210S-Y217Q, G097A-I111V-M124I-Y217Q,         G097A-I111V-M124V-P210S-Y217Q, G097A-N123Q-P210S-Y217Q,         G097A-N123Q-Y217Q, N061P-G097A-G128A-P210S-Y217Q,         N061P-G097A-G128S-Y217Q, N061P-G097A-I111V-M124V-Y217Q,         N061P-G097A-N123Q-Y217Q, N061P-G097A-S101N-I111V-M124V-Y217Q,         N061P-G097A-S101N-N123Q-Y217Q, N061P-G102A-P129S-Y217Q,         N061P-N062Q-G097A-G100N-S101N-Y217Q,         N061P-N062Q-G097A-G100N-Y217Q,         N061P-N062Q-G097A-G100Q-P201S-Y217Q,         N061P-N062Q-G097A-I111V-Y217Q,         N061P-N062Q-G097A-S101N-I111V-Y217Q,         N061P-S078N-G097A-I111V-M124I-Y217Q,         N061P-S078N-G102A-I111V-P129S-Y217Q,         N062Q-G097A-I111V-P210S-Y217Q, N062Q-G097A-I111V-Y217Q,         N062Q-S078N-G097A-I111V-Y217Q,         S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q,         S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,         S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q,         S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,         S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,         S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q,         S053G-N061P-G097A-G128S-Y217Q, S053G-N061P-G097A-M124I-Y217Q,         S053G-N061P-G097A-S101N-I111V-M124V-Y217Q,         S053G-N061P-G102A-P129S-P210S-Y217Q,         S053G-N061P-G102A-P129S-Y217Q,         S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q,         S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q,         S053G-N061P-S101N-G102A-P129S-Y217Q,         S053G-S078N-G097A-I111V-G128S-Y217Q, S078N-G097A-G128S-Y217Q,         S078N-G097A-I111V-M124V-Y217Q, N061P-G097A-M124I-Y217Q,         N061P-G097A-M124V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q,         N061P-N062Q-G097A-G100Q-S101N-Y217Q,         N061P-N062Q-G097A-G100Q-Y217Q, N061P-N062Q-G100N-G102A-Y217Q,         N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q,         S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q,         S053G-N061P-G097A-S101N-M124I-Y217Q,         S053G-N061P-G097A-S101N-N123Q-Y217Q,         N025G-S078N-G097A-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q,         S024G-G097A-S101N-G128A-Y217Q,         S024G-I035V-T055P-N061P-S078N-G097A-Y217Q,         S024G-N025G-N061P-S078N-G097A-S101N-G128A,         S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-N025G-N061P-S078N-S101N-G128A-Y217Q,         S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q,         S024G-N025G-S053G-N061P-G128A-Y217Q,         S024G-N025G-S053G-T055P-G097A-G128A-Y217Q,         S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,         S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q,         S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q,         S024G-N025G-S053G-T055P-S101N-G128A-Y217Q,         S024G-N025G-T055P-G097A-S101N-G128A-Y217Q,         S024G-N025G-T055P-N061P-S078N-G097A-Y217Q,         S024G-N061P-G097A-S101N-G128A-Y217Q,         S024G-S038G-S053G-S078N-S101N-G128A-Y217Q,         S024G-S053G-S078N-G097A-S101N-G128A-Y217Q,         S024G-S053G-S078N-S101N-G128A-Y217Q,         S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q,         S024G-T055P-G097A-G128A-Y217Q,         S024G-T055P-N061P-G097A-S101N-G128A,         S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,         S024G-T055P-S078N-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q,         T055P-N061P-G097A-A116S-G128A, T055P-N061P-S078N-G128A-Y217Q,         N025G-G097A-S101N-G128A-Y217Q,         N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,         N025G-S053G-N061P-S078N-G128A-Y217Q,         N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,         N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,         N025G-S078N-G097A-S101N-G128A-Y217Q,         N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         N025G-T055P-N061P-S078N-S101N-G128A-Y217Q,         N061P-S101N-G128A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q,         S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,         S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,         S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,         S024G-N025G-T055P-G097A-G128A-Y217Q,         S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,         S024G-S053G-N061P-G097A-G128A-Y217Q,         S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,         S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,         S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,         S024G-T055P-N061P-G097A-G128A-Y217Q,         S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,         S053G-N061P-S078N-G097A-G128A-Y217Q,         S053G-S078N-G097A-S101N-G128A-Y217Q,         S053G-T055P-G097A-S101N-G128A-Y217Q,         S053G-T055P-N061P-S101N-G128A-Y217Q,         S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,         T055P-G097A-S101N-G128A-Y217Q and         T055P-N061P-S078N-G097A-S101N-G128A-Y217Q;     -   (iii) a variant of a parent protease, said parent protease,         having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97%         or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant         comprising three, four, five or six of the following mutations         X24G/R, X53G, X78N, X101N, X128A/S and X217L/Q, said variant         optionally comprising at least one group of mutations selected         from the following groups of mutations: A001G, A001Y, A013G,         A013V, A015F, A015G, A015K, A015M, A015P, A015T, A015W, A015Y,         A029G, A073S, A088C, A088I, A088L, A088T, A088V, A098D, A098K,         A098P, A098R, A098W, A116D, A116E, A116R, A128S, A133L, A133M,         A133S, A134G, A134S, A137N, A137V, A144M, A144Q, A144S, A144T,         A144V, A151C, A176S, A176T, A179S, A216G, A216L, A216P, A216Q,         A216S, A216T, A216Y, A228T, A230C, A231C, A272I, A272L, A272Q,         A272S, A272T, A272W, A273S, A274G, A274L, A274Q, A274T, A274V,         D041E, D099G, D099N, D120A, D120K, D120Q, D120R, D120S, D140E,         D181S, D259E, E054D, E156D, E156T, E251V, F261G, F261H, F261L,         F261R, F261S, F261T, F261V, F261W, G007A, G007S, G020A, G020D,         G020S, G024N, G024R, G024S, G024T, G024V, G024W, G053H, G053K,         G053N, G053T, G097A, G097D, G097T, G131A, G131H, G131P, G131Q,         G131T, G131V, G160H, G160P, G166A, G166S, G166T, G211A, G211D,         G211M, G211N, G211P, G211Q, G211R, G211W, G215A, G215N, G215V,         H017L, H017M, H017T, H017V, H017Y, H039A, H039C, H039N, H226F,         H226I, H226M, H226S, H226V, H226Y, I035V, I079A, I079S, I079T,         I079V, I079W, I108V, I115L, I122A, I234L, I234V, K012R, K027R,         K136R, K141F, K213W, K237R, K256R, K265Q, L016A, L016F, L016I,         L016S, L016V, L042I, L075A, L075M, L075Q, L075V, L075Y, L082K,         L082M, L082Q, L082V, L090I, L196I, L196V, L209Q, L209W, L233A,         L233Q, L233V, L235I, L235K, L250I, L257A, L257H, L257Q, L257S,         L257Y, L267Q, L267R, L267S, L267T, M199V, N025F, N025G, N025H,         N025K, N025L, N025M, N025S, N025Y, N061F, N061H, N061P, N061S,         N061T, N061V, N061W, N076G, N076W, N078S, N078T, N078V, N101A,         N101H, N101L, N101T, N109K, N109R, N117A, N117E, N117H, N117K,         N118G, N184G, N184H, N184I, N184S, N184V, N212A, N212F, N212I,         N212K, N212P, N212Q, N212S, N212Y, N218A, N218H, N218L, N218S,         N240E, N240H, N240L, N240R, N240T, N243A, N243Q, N243T, N243V,         N252A, N252K, N252L, N252Q, N252R, N252S, N252T, N269Q, N269S,         P014G, P014Q, P014T, P040A, P040H, P040S, P040T, P040V, P040Y,         P086A, P086C, P086F, P086H, P086S, P129D, P129G, P129K, P129T,         P172A, P172Q, P194A, P194G, P194L, P194M, P194S, P194V, P194Y,         P210G, P210R, P210V, Q002A, Q002S, Q010A, Q010F, Q010H, Q010I,         Q010L, Q010N, Q010S, Q010T, Q019A, Q019G, Q019N, Q019S, Q019T,         Q019V, Q019W, Q059I, Q103L, Q103S, Q185A, Q185H, Q185L, Q185T,         Q185Y, Q206P, Q206S, Q206Y, Q217I, Q217N, Q217S, Q217T, Q245K,         Q275D, Q275S, Q275W, S003A, S003G, S003H, S003M, S003P, S003Q,         S003T, S003V, S009I, S009L, S009P, S009T, S009W, S018A, S018G,         S018I, S018L, S018M, S018N, S018P, S018V, S018W, S033T, S037Q,         S037T, S037V, S038G, S038H, S038K, S038Q, S038T, S063K, S063N,         S063Q, S063T, S087A, S087F, S087G, S087Q, S087T, S089L, S089M,         S089N, S089Q, S089T, S089W, S130A, S130F, S130G, S130L, S130V,         S145A, S145H, S145M, S145V, S159A, S159G, S159H, S159Q, S159R,         S161A, S161G, S161H, S161L, S161M, S161P, S161Q, S161W, S162A,         S162F, S162G, S162L, S162N, S162P, S162R, S162V, S163P, S173A,         S173G, S182A, S182H, S182K, S182L, S182N, S182P, S182Q, S182T,         S183A, S183G, S183H, S183Q, S188A, S188G, S188T, S188V, S191A,         S204A, S204I, S204L, S204Q, S204V, S224C, S236A, S236N, S236Q,         S248A, S248F, S248G, S248I, S248K, S248L, S248M, S248N, S248Q,         S248T, S248V, S249A, S249C, S249H, S249Q, S249T, S249W, S249Y,         S260H, S260N, S260P, S260T, T022H, T022K, T022N, T022R, T022S,         T022Y, T055A, T055G, T055L, T055N, T055P, T055Q, T071S, T158H,         T158S, T164N, T208C, T208L, T220S, T242N, T244A, T244G, T244H,         T244I, T244Q, T244S, T244V, T244W, T253A, T253G, T253H, T253Q,         T254V, T255A, T255G, T255H, T255I, T255Q, T255Y, V004G, V004N,         V004R, V008A, V008C, V008M, V026I, V044I, V044L, V045H, V045K,         V045L, V045M, V045Q, V045S, V045W, V045Y, V051I, V081L, V081Q,         V081T, V084A, V084S, V084T, V093I, V121I, V143N, V143S, V143Y,         V147C, V147I, V147L, V147T, V180I, V180L, V180T, V192A, V192S,         V192T, V198I, V198L, V198M, V203H, V203I, V203L, V203N, V203Q,         V203T, V203W, V203Y, V270A, V270S, V270T, W241M, W241Y, Y006G,         Y006H, Y006I, Y006K, Y006L, Y006P, Y006Q, Y006T, Y006V, Y006W,         Y021A, Y021D, Y021E, Y021L, Y021Q, Y021R, Y021S, Y104F, Y104I,         Y214L, Y214V, Y214W, Y262A, Y262G, Y262L, Y262N, Y262S, Y262W,         Y263G, Y263W, A001F, A001K, A001L, A001M, A001Q, A001R, A001S,         A001T, A001V, A013C, A013S, A015D, A015E, A015L, A015R, A048S,         A073N, A073T, A074G, A074S, A085C, A085G, A085S, A085V, A088M,         A088S, A092S, A098G, A114G, A133P, A137E, A137H, A144G, A144H,         A144K, A144L, A144N, A153S, A153V, A176C, A179G, A187G, A187S,         A200G, A216W, A223S, A228S, A230T, A230V, A231V, A232C, A232V,         A272E, A272G, A272K, A272P, A273G, A273L, A273V, A274M, A274R,         D036E, D099A, D099Q, D120E, D181A, D181G, D259A, D259G, D259Q,         D259T, E156A, E156S, E251I, E251L, E251Q, E251T, F058Y, F261C,         F261D, F261K, F261P, G020E, G020F, G020H, G020L, G020N, G020Q,         G020R, G020T, G020Y, G024A, G024P, G053A, G053D, G053E, G053F,         G053L, G053Q, G053S, G053Y, G097K, G097M, G157A, G157S, G160A,         G160L, G166C, G166I, G166Q, G169A, G211K, G215H, G215L, G215S,         G215T, G215W, G258S, H017I, H039S, H226L, H238N, H238Y, I011L,         I011V, I031L, I079F, I079K, I079L, I079M, I079Q, I205A, I205V,         I268L, I268M, K012G, K043F, K043H, K043I, K043N, K043Q, K043T,         K141A, K141R, K141W, K170A, K213A, K213G, K213H, K213I, K213L,         K213N, K213Q, K213R, K213S, K213T, K213V, K237A, K237H, K237I,         K237L, K237N, K237S, K256A, K256G, K256H, K256M, K256P, K256Q,         K256W, K265H, L016E, L042V, L075G, L075H, L075I, L075T, L082A,         L082F, L082H, L082R, L082S, L082T, L090M, L135F, L196M, L209C,         L209H, L209S, L233S, L235M, L235R, L235W, L257C, L257G, L267F,         M050Y, M119C, M119I, M124L, N025C, N025E, N025P, N061A, N061G,         N061I, N061K, N061L, N061Q, N061R, N062S, N062T, N076A, N076P,         N076Q, N076S, N076T, N076V, N078G, N078H, N078K, N078P, N078Q,         N078R, N101F, N117R, N117S, N118D, N118H, N118Q, N118R, N118S,         N118T, N184C, N184E, N184R, N212D, N212R, N212W, N218F, N218G,         N218M, N218P, N218T, N218V, N218W, N240A, N240G, N240Q, N240S,         N240W, N243C, N243G, N243S, N252V, N269H, P005A, P005D, P005M,         P005Q, P014A, P014M, P014R, P014V, P040F, P040R, P040W, P129E,         P129R, P172E, P172K, P194H, P194R, P194W, P201A, P201G, P210L,         P239K, P239R, Q002D, Q002E, Q002G, Q002I, Q002P, Q002V, Q010D,         Q010R, Q019C, Q019D, Q019E, Q019H, Q019L, Q019P, Q019R, Q059A,         Q059E, Q059L, Q059S, Q059T, Q103W, Q185D, Q185K, Q185R, Q185W,         Q206G, Q206H, Q206L, Q206V, Q206W, Q217E, Q217F, Q217H, Q217L,         Q217V, Q245M, Q271A, Q271D, Q271G, Q271L, Q271P, Q271T, Q271Y,         Q275F, Q275L, Q275P, Q275R, S003D, S003F, S063A, S063F, S063G,         S063M, S063R, S063Y, S087C, S087K, S087L, S087M, S087N, S087Y,         S089A, S089D, S089F, S089G, S089H, S089I, S089K, S089R, S089V,         S089Y, S130D, S130E, S130K, S130W, S145G, S145L, S145R, S145T,         S159D, S159L, S159W, S161E, S161R, S162C, S162E, S162W, S163A,         S182E, S182R, S183C, S183D, S183P, S183R, S188D, S188P, S204G,         S204Y, S207G, S224G, S224T, S236C, S236G, S248D, S248H, S248R,         S249E, S249L, S249R, S260A, S260G, S260K, S260Q, S260V, S260Y,         T022L, T055D, T055E, T055I, T055K, T055M, T055S, T055V, T055Y,         T158A, T158G, T158L, T158Q, T158V, T164K, T164Q, T208S, T244D,         T244E, T244R, T253E, T253R, T253Y, T254G, T255D, T255E, T255K,         T255R, V026A, V028I, V028L, V030I, V044C, V044P, V045E, V045G,         V045N, V072L, V081A, V081G, V081H, V081S, V084I, V084M, V095A,         V095C, V143A, V143F, V143H, V143Q, V143T, V143W, V147A, V147Q,         V147S, V148I, V148L, V149C, V149I, V149L, V165L, V180A, V180C,         V180M, V192C, V192F, V192I, V192Q, V192Y, V203A, V203G, V203K,         V203S, V270C, V270L, V270P, W241F, Y006A, Y006M, Y006N, Y006R,         Y006S, Y021C, Y091W, Y104V, Y104W, Y262C, Y262D, Y262E, Y262H,         Y262I, Y262R, Y262V, A088T-A116T, A088T-N109G, A088T-N243V,         A088T-S249A, A116T, A116T-G131H, A116T-N243V, A116T-S162G,         A116T-T158S, A128S-K256R, A128S-L257G, A128S-S248N, A128S-T158S,         G024E-A116T, G024E-K256R, G024E-L257G, G024E-N243V, G024E-T158S,         G131H-S249A, K043Y-A088T, N076D-K256R, N109G, N109G-A116T,         N109G-A128S, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A,         N109G-L257G, N109G-N243V, N243V-K256R, N243V-S248N, N243V-S249A,         Q103H-A128S, Q103H-G131H, Q103H-K256R, Q103H-L257G, Q103H-N243V,         Q103H-S248N, Q103H-S249A, Q103H-T158S, S033T-N243V, S033T-T158S,         S063G-A128S, S063G-K256R, S063G-S162G, S063G-T158S, S248N-L257G,         S249A, T158S-S249A, A001E-A128S-G131H-N243V,         A001E-G131H-G169A-N243V, A001E-N109G, A001E-S033T-N109G-N218S,         A088T-A128S, A116T-G169A, A116T-Q206D, A128S-N243V, G024E,         G024E-A088T, G024E-A128S, G024E-G131H, G024E-N218S, G024E-S162G,         G024E-S249A, G169A, G169A-K256R, G169A-L257G, G169A-N218S,         G169A-N243V, G169A-Q206D, G169A-S248N, G169A-S249A, K043Y,         K043Y-A116T, K043Y-G169A, N076D, N109G-G169A, N218S-L257G,         P040E-N109G-A128S-G131H, Q206D-L257G, Q206D-N243V, S033T-A128S,         S033T-A128S-G131H-N243P, S033T-K256R, S033T-L257G, S033T-N076D,         S033T-N109G, S033T-N218S, S033T-P040E-Q103H-N109G,         S033T-Q103H-A128S-G131H, S033T-S063G, S033T-S248N,         S063G-N109G-A128S-G131H, S063G-N243V, S063G-Q206D, S248N-K256R,         T158S-Q206D, A001E-A128S, A001E-G131H, A001E-K256R, A001E-N218S,         A001E-N243V, A001E-S033T, A001E-S063G, A001E-S162G, A088T-K256R,         A088T-N218S, A088T-Q103H, A088T-S162G,         A088T-T158SA116T-A128SA116T-G131HA116T-K256R, A116T-L257G,         A116T-S162G, A116T-S248N, A116T-S249A, A128S-G169A, A128S-N218S,         A128S-S162G, A128S-S249A, G024E, G024E-N109G, G024E-Q103H,         G024E-S033T, G024E-S063G, G024E-S248N, G131H-L257G, G131H-N243V,         G131H-S162G, G131H-T158S, G169A, G169A-L257G, G169A-S248N,         K043Y-A128S, K043Y-G131H, K043Y-K256R, K043Y-L257G, K043Y-N109G,         K043Y-N243V, K043Y-Q103H, K043Y-S063G, K043Y-S162G, K043Y-S248N,         K043Y-S249A, K043Y-T158S, K256R-L257G, N061G-N109G-N243V,         N061S-A128S-N243V-S260P, N061S-N109G-A128S-N243V-S260P,         N061S-N109G-A128S-S260P, N076D-A088T, N076D-A128S, N076D-G169A,         N076D-N218S, N076D-N243V, N076D-S162G, N076D-S248N, N076D-T158S,         N109A-A128S-N243V-K256R, N109G-A128S-G131H-N243V-S248N-K256R,         N109G-A128S-N243V-S248A-K256R,         N109G-A128S-N243V-S248N-K256R-L257G,         N109G-A128S-S162G-N243V-S248N-K256R,         N109G-A128S-T158S-N243V-S248N-K256R, N109G-Q206D, N109G-S162G,         N109G-S249A, N109G-T158S, N109Q-A128S-N243V-K256R,         N109S-A128S-N243V-K256R, N218S-K256R, N218S-N243V,         P040A-N109G-A128S-N243V-S248N-K256R, Q103H, Q103H-A116T,         Q103H-G169A, Q103H-N109G, Q103H-N218S, Q103H-S162G,         S009T-A128S-K141R-N243V, S009T-N109G-A128S-K141R,         S009T-N109G-A128S-K141R-N243V,         S009T-S018T-Y021N-N109G-A128S-K141R, S018T-Y021N-N109G-A128S,         S018T-Y021N-N109G-A128S-N243V,         S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S033T-A088T,         S033T-A116T, S033T-G131H, S033T-K043Y, S033T-L257G, S033T-N109G,         S033T-Q103H, S033T-Q206D, S033T-S162G, S033T-S249A, S063G,         S063G-A088T, S063G-A116T, S063G-L257G, S063G-N076D, S063G-N109G,         S063G-N218S, S063G-Q103H, S063G-S248N, S063G-S249A, S162G-G169A,         S162G-L257G, S162G-N218S, S162G-N243V, S162G-S248N, S162G-S249A,         S248N-S249A, S249A-K256R, S249A-L257G, T158S-G169A, T158S-K256R,         T158S-L257G, T158S-N218S, T158S-S248N, A001E, A001E-A088T,         A001E-A116T, A001E-G169A, A001E-L257G, A001E-N109G,         A001E-S033T-N109G-N243V, A001E-T158S, A088T-G169A, A088T-Q206D,         A116T-N218S, A128S-G131H, A128S-N243V-S248N-K256R, A128S-Q206D,         G024E-K043Y, G024E-N076D, G024E-Q206D, G131H-G169A, G131H-N218S,         G131H-N243V-K256R, G131H-Q206D, G131H-S248N, G169A-K256R,         G169A-N218S, G169A-N243V, G169A-Q206D, K043Y-N076D, K043Y-N218S,         N061P-N109G-G131H-N243V, N061P-N109G-N243V,         N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-N243V,         N076D, N076D-G131H, N076D-L257G, N076D-N109G, N076D-Q103H,         N076D-S249A, N109G-A128S-N243V-S248N,         N109G-A128S-N243V-S248N-K256R, N109G-A128S-S248N-K256R,         N109G-N243P-S248A-K256R, N109G-N243P-S248N-K256R,         N109G-N243V-K256R, N109G-N243V-S248A-K256R, N109G-N243V-S248N,         N109G-N243V-S248N-K256R, N109G-S248N-K256R, N218S-S249A,         N243V-S248N-K256R, Q103H-Q206D, Q206D, Q206D-K256R, Q206D-N218S,         Q206D-S248N, Q206D-S249A,         S009T-N109G-A128S-K141R-N243V-S248N-K256R,         S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V,         S018T-Y021N-N061S-A128S-N243V-S260P,         S018T-Y021N-N061S-N109G-A128S-S260P,         S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T,         S033T-G169A, S033T-N076D-A128S-N218S,         S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R,         S033T-N109G-A128S-N243P-S248N-K256R,         S033T-N109G-A128S-N243V-S248N-K256R, S063G-G131H, S063G-G169A,         S162G-Q206D, T158S-S162G, A088T-A116T-G131H-L257G,         A088T-A116T-G131H-N218S-A274T, A088T-A116T-G131H-N218S-K256R,         A088T-A116T-G131H-N218S-K256R-L257G,         A088T-A116T-G131H-N218S-N243V,         A088T-A116T-G131H-N218S-N243V-L257G,         A088T-A116T-G131H-N218S-N243V-S248N-K256R,         A088T-A116T-G131H-N218S-N243V-S248N-K256R-L257G,         A088T-A116T-G131H-N218S-N243V-S248N-L257G,         A088T-A116T-G131H-N218S-S248N,         A088T-A116T-G131H-N218S-S248N-L257G, A088T-A116T-G131H-N243V,         A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-N243V-S248N,         A088T-A116T-G131H-N243V-S248N-A274V,         A088T-A116T-G131H-N243V-S248N-K256R-L257G,         A088T-A116T-G131H-N243V-S248N-K256R-L257G,         A088T-A116T-G131H-N243V-S248N-L257G,         A088T-A116T-G131H-S248N-K256R,         A088T-A116T-G131H-S248N-K256R-L257G,         A088T-A116T-G131H-T158S-K256R,         A088T-A116T-G131H-T158S-K256R-L257G,         A088T-A116T-G131H-T158S-N218S-K256R,         A088T-A116T-G131H-T158S-N218S-K256R-L257G,         A088T-A116T-G131H-T158S-N218S-L257G,         A088T-A116T-G131H-T158S-N218S-N243V-K256R,         A088T-A116T-G131H-T158S-N218S-N243V-K256R-L257G,         A088T-A116T-G131H-T158S-N218S-N243V-L257G,         A088T-A116T-G131H-T158S-N218S-N243V-S248N,         A088T-A116T-G131H-T158S-N218S-N243V-S248N,         A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R,         A088T-A116T-G131H-T158S-N243V-K256R,         A088T-A116T-G131H-T158S-N243V-K256R,         A088T-A116T-G131H-T158S-N243V-K256R-L257G,         A088T-A116T-G131H-T158S-N243V-L257G,         A088T-A116T-G131H-T158S-N243V-S248N,         A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G,         A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G,         A088T-A116T-G131H-T158S-N243V-S248N-L257G,         A088T-A116T-G131H-T158S-S248N-K256R,         A088T-A116T-G131H-T158S-S248N-K256R-L257G, A088T-A116T-K256R,         A088T-A116T-L257G, A088T-A116T-N218S,         A088T-A116T-N218S-N243V-K256R, A088T-A116T-N218S-N243V-N269D,         A088T-A116T-N218S-N243V-S248N,         A088T-A116T-N218S-N243V-S248N-K256R-L257G,         A088T-A116T-N218S-N243V-S248N-L257G, A088T-A116T-N218S-S248N,         A088T-A116T-N218S-S248N-L257G, A088T-A116T-N243V-K256R-L257G,         A088T-A116T-N243V-S248N-K256R, A088T-A116T-S248N,         A088T-A116T-S248N-K256R,         A088T-A116T-T158S-A216S-N218S-N243V-K256R-L257G,         A088T-A116T-T158S-N218S-K256R,         A088T-A116T-T158S-N218S-N243V-K256R,         A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N243V-K256R,         A088T-A116T-T158S-N243V-S248N,         A088T-A116T-T158S-N243V-S248N-K256R,         A088T-A116T-T158S-N243V-S248N-L257G, A088T-A116T-T158S-S248N,         A088T-G131D-T158S-N243V-S248N, A088T-G131H-A138V-N218S-L257G,         A088T-G131H-K256R, A088T-G131H-N218S-N243V-K256R,         A088T-G131H-N218S-N243V-K256R, A088T-G131H-N218S-S248N,         A088T-G131H-N218S-S248N-K256R-L257G, A088T-G131H-N218T-L257G,         A088T-G131H-N243V-L257G, A088T-G131H-N243V-S248N-K256R,         A088T-G131H-S248N, A088T-G131H-S248N-L257G,         A088T-G131H-T158S-K256R, A088T-G131H-T158S-K256R-L257G,         A088T-G131H-T158S-N218S, A088T-G131H-T158S-N218S,         A088T-G131H-T158S-N218S-K256R-L257G,         A088T-G131H-T158S-N218S-N243V-K256R,         A088T-G131H-T158S-N218S-N243V-K256R,         A088T-G131H-T158S-N218S-N243V-S248N,         A088T-G131H-T158S-N218S-S248N,         A088T-G131H-T158S-N218S-S248N-K256R,         A088T-G131H-T158S-N218S-S248N-L257G-I268V,         A088T-G131H-T158S-N243V-K256R,         A088T-G131H-T158S-N243V-K256R-L257G,         A088T-G131H-T158S-N243V-S248N,         A088T-G131H-T158S-N243V-S248N-K256R-L257G,         A088T-G131H-T158S-S248N, A088T-G131H-T158S-S248N-K256R,         A088T-N109G-A116T-G131H-K256R,         A088T-N109G-A116T-G131H-K256R-L257G,         A088T-N109G-A116T-G131H-K256R-L257G,         A088T-N109G-A116T-G131H-N218S-K256R,         A088T-N109G-A116T-G131H-N218S-K256R,         A088T-N109G-A116T-G131H-N218S-K256R-L257G,         A088T-N109G-A116T-G131H-N218S-N243V-L257G,         A088T-N109G-A116T-G131H-N218S-S248N,         A088T-N109G-A116T-G131H-N218S-S248N-L257G,         A088T-N109G-A116T-G131H-N243V-K256R,         A088T-N109G-A116T-G131H-N243V-K256R-L257G,         A088T-N109G-A116T-G131H-N243V-S248N-K256R,         A088T-N109G-A116T-G131H-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-S248N-L257G,         A088T-N109G-A116T-G131H-T158S-K256R,         A088T-N109G-A116T-G131H-T158S-L257G,         A088T-N109G-A116T-G131H-T158S-N218S,         A088T-N109G-A116T-G131H-T158S-N218S-L257G,         A088T-N109G-A116T-G131H-T158S-N218S-N243V,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-L257G,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-T158S-N218S-S248N,         A088T-N109G-A116T-G131H-T158S-N218T-K256R,         A088T-N109G-A116T-G131H-T158S-N243V,         A088T-N109G-A116T-G131H-T158S-N243V-S248N,         A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G,         A088T-N109G-A116T-G131H-V149A-T158S-N218S-K256R,         A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R,         A088T-N109G-A116T-N218S-N243V,         A088T-N109G-A116T-N218S-N243V-K256R-L257G,         A088T-N109G-A116T-N218S-N243V-L257G,         A088T-N109G-A116T-N218S-N243V-S248N-K256R,         A088T-N109G-A116T-N218S-S248N,         A088T-N109G-A116T-N218S-S248N-K256R-L257G,         A088T-N109G-A116T-N243V-K256R,         A088T-N109G-A116T-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-N243V-S248N-L257G, A088T-N109G-A116T-S248N,         A088T-N109G-A116T-S248N-K256R-L257G,         A088T-N109G-A116T-S248N-L257G, A088T-N109G-A116T-T158S,         A088T-N109G-A116T-T158S-K256R, A088T-N109G-A116T-T158S-N218S,         A088T-N109G-A116T-T158S-N218S-L257G,         A088T-N109G-A116T-T158S-N218S-N243V-S248N,         A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,         A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-T158S-N218S-S248N,         A088T-N109G-A116T-T158S-N218S-S248N-K256R,         A088T-N109G-A116T-T158S-N218S-S248N-L257G,         A088T-N109G-A116T-T158S-N243V-K256R,         A088T-N109G-A116T-T158S-N243V-S248N,         A088T-N109G-A116T-T158S-S248N.         A088T-N109G-A116T-T158S-S248N-K256R,         A088T-N109G-A137E-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-G131H-A152S-T158S-N218S-S248N-K256R,         A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S,         A088T-N109G-G131H-N218S, A088T-N109G-G131H-N218S-K256R,         A088T-N109G-G131H-N218S-L257G, A088T-N109G-G131H-N218S-N243V,         A088T-N109G-G131H-N218S-N243V-K256R,         A088T-N109G-G131H-N218S-N243V-K256R-L257G,         A088T-N109G-G131H-N218S-N243V-S248N-K256R,         A088T-N109G-G131H-N218S-S248N,         A088T-N109G-G131H-N218S-S248N-K256R,         A088T-N109G-G131H-N218S-S248N-K256R-L257G,         A088T-N109G-G131H-N243V-K256R,         A088T-N109G-G131H-N243V-K256R-L257G,         A088T-N109G-G131H-N243V-L257G,         A088T-N109G-G131H-N243V-S248N-K256R,         A088T-N109G-G131H-S248N-K256R, A088T-N109G-G131H-S248N-L257G,         A088T-N109G-G131H-T158S, A088T-N109G-G131H-T158S-K256R,         A088T-N109G-G131H-T158S-K256R-L257G,         A088T-N109G-G131H-T158S-N218S-K256R,         A088T-N109G-G131H-T158S-N218S-L257G,         A088T-N109G-G131H-T158S-N218S-N243V,         A088T-N109G-G131H-T158S-N218S-N243V-K256R,         A088T-N109G-G131H-T158S-N218S-N243V-S248N,         A088T-N109G-G131H-T158S-N218S-S248N-L257G,         A088T-N109G-G131H-T158S-N243V-K256R-L257G,         A088T-N109G-G131H-T158S-N243V-S248N,         A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G,         A088T-N109G-G131H-T158S-N243V-S248N-L257G,         A088T-N109G-G131H-T158S-S248N,         A088T-N109G-G131H-T158S-S248N-L257G,         A088T-N109G-G131H-V149A-K256R-L257G,         A088T-N109G-G154A-N155P-E156T-G157L-T158M-S159E-G160E-S161L,         A088T-N109G-K256R-L257G, A088T-N109G-N218S-K256R,         A088T-N109G-N218S-N243V-L257G,         A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-S248N,         A088T-N109G-N218S-S248N-L257G, A088T-N109G-N243V-S248N-K256R,         A088T-N109G-S248N, A088T-N109G-T158S-N218S,         A088T-N109G-T158S-N218S-K256R-Q271H,         A088T-N109G-T158S-N218S-N243V,         A088T-N109G-T158S-N218S-N243V-K256R-Q275R,         A088T-N109G-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-T158S-N218S-S248N,         A088T-N109G-T158S-N218S-S248N-K256R,         A088T-N109G-T158S-N218S-S248N-N269D,         A088T-N109G-T158S-N243V-K256R,         A088T-N109G-T158S-N243V-S248N-K256R,         A088T-N109G-T158S-N243V-S248N-K256R-L257G-N269D,         A088T-N109G-T158S-N243V-S248N-L257G,         A088T-N109G-T158S-S248N-K256R-L257G,         A088T-N109G-T158S-S248N-L257G,         A088T-N109G-V147A-N218S-N243V-K256R, A088T-N218S-K256R,         A088T-N218S-L257G-I268V, A088T-N218S-N243V,         A088T-N218S-N243V-K256R, A088T-N218S-N243V-K256R-L257G,         A088T-N218S-N243V-L257G, A088T-N218S-N243V-S248N-K256R-L257G,         A088T-N218S-N243V-S248N-L257G, A088T-N218S-N243V-S248N-N269S,         A088T-N218S-S248N-K256R, A088T-N243V-S248N,         A088T-N243V-S248N-K256R, A088T-N243V-S248N-K256R-L257G,         A088T-N243V-S248N-L257G, A088T-S248N, A088T-S248N-K256R-L257G,         A088T-S248N-L257G, A088T-S248N-L257G-I268V, A088T-T158S-N218S,         A088T-T158S-N218S-K256R, A088T-T158S-N218S-L257G,         A088T-T158S-N218S-N243V-K256R-I268V,         A088T-T158S-N218S-N243V-K256R-L257G,         A088T-T158S-N218S-N243V-S248N-L257G, A088T-T158S-N218S-S248N,         A088T-T158S-N243V-K256R, A088T-T158S-N243V-K256R-L257G,         A088T-T158S-N243V-S248N-K256R, A088T-T158S-N243V-S248N-L257G,         A088T-T158S-S248N, A088T-T158S-S248N-L257G, A088T-V147A-K256R,         A098S-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         A116T-G131H-N218S-K256R, A116T-G131H-N218S-K256R-L257G,         A116T-G131H-N218S-L257G, A116T-G131H-N218S-N243V-S248N-L257G,         A116T-G131H-N218S-S248N-K256R-L257G, A116T-G131H-N243V-S248N,         A116T-G131H-N243V-S248N-L257G,         A116T-G131H-T158S-A231V-N243V-L257G, A116T-G131H-T158S-K256R,         A116T-G131H-T158S-K256R-L257G, A116T-G131H-T158S-N218S-K256R,         A116T-G131H-T158S-N218S-K256R-L257G,         A116T-G131H-T158S-N218S-N243V,         A116T-G131H-T158S-N218S-N243V-K256R,         A116T-G131H-T158S-N218S-N243V-K256R-L257G,         A116T-G131H-T158S-N218S-N243V-L257G,         A116T-G131H-T158S-N218S-N243V-S248N-K256R,         A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         A116T-G131H-T158S-N218S-N243V-S248N-L257G,         A116T-G131H-T158S-N218S-S248N-K256R,         A116T-G131H-T158S-N218T-L257G, A116T-G131H-T158S-S248N-K256R,         A116T-G131H-T158S-S248N-L257G, A116T-N218S-K256R,         A116T-N218S-K256R-L257G, A116T-N218S-N243V-S248N-K256R,         A116T-N218S-N243V-S248N-K256R-L257G,         A116T-N218S-N243V-S248N-L257G, A116T-N218S-S248N,         A116T-N218S-S248N-K256R, A116T-N218S-S248N-L257G,         A116T-N243V-S248N-L257G, A116T-S248N-L257G,         A116T-T158S-L257G-Q271R, A116T-T158S-N218S-L257G,         A116T-T158S-N218S-N243V-K256R-L257G,         A116T-T158S-N218S-S248N-K256R,         A116T-T158S-N218S-S248N-K256R-L257G,         A116T-T158S-N243V-S248N-K256R,         A116T-T158S-N243V-S248N-K256R-L257G,         G024S-G053S-N078S-G097A-N101S-A128S, G131H-N218S,         G131H-N218S-K256R, G131H-N218S-N243V-K256R,         G131H-N218S-N243V-K256R-L257G, G131H-N218S-N243V-S248N,         G131H-N218S-N243V-S248N-K256R, G131H-N218S-S248N-K256R-L257G,         G131H-N218S-S248N-L257G, G131H-N243V-K256R-L257G,         G131H-N243V-S248N-K256R-L257G, G131H-S248N-K256R,         G131H-T158S-K256R, G131H-T158S-K256R-L257G,         G131H-T158S-N218S-K256R, G131H-T158S-N218S-K256R-L257G,         G131H-T158S-N218S-N243V-K256R-L257G,         G131H-T158S-N218S-N243V-S248N,         G131H-T158S-N218S-S248N-K256R-L257G,         G131H-T158S-N218S-S248N-L257G, G131H-T158S-N243V-K256R,         G131H-T158S-N243V-S248N-K256R, G131H-T158S-S248N-L257G,         G131H-V147I-N218S-S248N-K256R,         I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         I107T-N109G-G131H-N218S-N243V-K256R-L257G, N109G,         N109G-A116T-G131H,         N109G-A116T-G131H-A144V-T158S-S248N-K256R-L257G,         N109G-A116T-G131H-K256R-L257G, N109G-A116T-G131H-L257G,         N109G-A116T-G131H-N218S-N243V-K256R,         N109G-A116T-G131H-N218S-N243V-S248N-K256R,         N109G-A116T-G131H-N243V, N109G-A116T-G131H-N243V-K256R-L257G,         N109G-A116T-G131H-N243V-S248N,         N109G-A116T-G131H-N243V-S248N-K256R,         N109G-A116T-G131H-S248N-K256R, N109G-A116T-G131H-T158S-K256R,         N109G-A116T-G131H-T158S-K256R-L257G,         N109G-A116T-G131H-T158S-L257G, N109G-A116T-G131H-T158S-N218S,         N109G-A116T-G131H-T158S-N218S-K256R-L257G,         N109G-A116T-G131H-T158S-N218S-L257G,         N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         N109G-A116T-G131H-T158S-N218S-S248N-K256R,         N109G-A116T-G131H-T158S-N218S-S248N-K256R-L257G,         N109G-A116T-G131H-T158S-N243V-L257G,         N109G-A116T-G131H-T158S-S248N-L257G,         N109G-A116T-G131H-V147A-T158S-N218S-K256R-L257G,         N109G-A116T-G131H-V149A-T158S-N218S-N243V-S248N-L257G,         N109G-A116T-K256R, N109G-A116T-N218S-K256R,         N109G-A116T-N218S-N243V, N109G-A116T-N218S-N243V-L257G,         N109G-A116T-N218S-N243V-S248N-I268V,         N109G-A116T-N218S-N243V-S248N-K256R-L257G,         N109G-A116T-N218S-N243V-S248N-L257G, N109G-A116T-N243V-S248N,         N109G-A116T-N243V-S248N-K256R, N109G-A116T-N243V-S248N-L257G,         N109G-A116T-S248N-K256R, N109G-A116T-T158S,         N109G-A116T-T158S-N218S-N243V-S248N-K256R,         N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G,         N109G-A116T-T158S-N218S-S248N-K256R,         N109G-A116T-T158S-N243V-L257G, N109G-A116T-T158S-N243V-S248N,         N109G-A116T-T158S-S248N, N109G-A116T-T158S-S248N-K256R,         N109G-G131H, N109G-G131H-K256R, N109G-G131H-N218S-K256R,         N109G-G131H-N218S-K256R-L257G, N109G-G131H-N218S-N243V-L257G,         N109G-G131H-N218S-N243V-S248N-K256R,         N109G-G131H-N218S-N243V-S248N-K256R-L257G,         N109G-G131H-N218S-N243V-S248N-L257G,         N109G-G131H-N218S-S248N-L257G, N109G-G131H-N243V,         N109G-G131H-N243V-K256R, N109G-G131H-N243V-S248N,         N109G-G131H-N243V-S248N-K256R-L257G,         N109G-G131H-N243V-S248N-L257G,         N109G-G131H-T158S-N218S-K256R-L257G,         N109G-G131H-T158S-N218S-L257G, N109G-G131H-T158S-N218S-N243V,         N109G-G131H-T158S-N218S-N243V-K256R-L257G,         N109G-G131H-T158S-N218S-N243V-S248N,         N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         N109G-G131H-T158S-N218S-N243V-S248N-L257G,         N109G-G131H-T158S-N218S-S248N-K256R-L257G,         N109G-G131H-T158S-N243V-K256R-I268V,         N109G-G131H-T158S-N243V-S248N,         N109G-G131H-T158S-N243V-S248N-K256R,         N109G-G131H-T158S-N243V-S248N-L257G, N109G-G131H-T158S-S248N,         N109G-G131H-T158S-S248N-K256R-L257G,         N109G-K141E-N218S-S248N-L257G, N109G-N218S,         N109G-N218S-N243V-K256R, N109G-N218S-N243V-L257G,         N109G-N218S-N243V-S248N-S260F, N109G-N218S-S248N,         N109G-N218S-S248N-K256R, N109G-N243V-S248N-L257G,         N109G-N243V-S248N-L257G-Q275R,         N109G-S182F-S204F-S207L-N218S-S236F-S248N-L257G,         N109G-T158S-K256R-L257G, N109G-T158S-L257G,         N109G-T158S-N218S-N243V-K256R, N109G-T158S-N218S-N243V-S248N,         N109G-T158S-N218S-N243V-S248N-L257G, N109G-T158S-N243V-K256R,         N109G-T158S-N243V-S248N-K256R, N109G-T158S-N243V-S248N-L257G,         N109G-T158S-S248N-L257G, N218S-N243V-L257G,         N218S-N243V-S248N-K256R, N243V-K256R-L257G,         N243V-S248N-L257G-Q271R,         P057Q-A088T-N109G-A116T-G131H-T158S-N218S-S248N,         S003P-A116T-N218S-K256R,         S003P-N109G-G131H-N218S-N243V-S248N-K256R-L257G,         S248N-K256R-L257G, T158S-K256R-L257G, T158S-N218S-A272V,         T158S-N218S-K256R-L257G, T158S-N218S-L233S, T158S-N218S-N243V,         T158S-N218S-N243V-K256R-L257G, T158S-N218S-N243V-L257G,         T158S-N218S-N243V-S248N-K256R, T158S-N218S-S248N-K256R,         T158S-N243V, T158S-N243V-K256R-L257G, T158S-N243V-S248N,         T158S-N243V-S248N-K256R-N269D,         V004A-N109G-A116T-T158S-N218S-S248N-L257G,         A088T-A098S-N218S-K256R, A088T-A116T-G131H-K256R,         A088T-A116T-G131H-K256R-L257G-L267M,         A088T-A116T-G131H-N218S-N243V-K256R,         A088T-A116T-G131H-N218S-N243V-K256R-L257G,         A088T-A116T-G131H-N218S-N243V-S248N,         A088T-A116T-G131H-N218S-S248N,         A088T-A116T-G131H-N218S-S248N-K256R,         A088T-A116T-G131H-N218S-S248N-K256R, A088T-A116T-G131H-N243V,         A088T-A116T-G131H-S248N, A088T-A116T-G131H-S248N-L257G,         A088T-A116T-G131H-S248N-L257G, A088T-A116T-G131H-T158S-N218S,         A088T-A116T-G131H-T158S-N218S-K256R-L257G,         A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R,         A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,         A088T-A116T-G131H-T158S-N218S-S248N,         A088T-A116T-G131H-T158S-N218S-S248N-K256R,         A088T-A116T-K256R-L257G, A088T-A116T-N218S-I268V,         A088T-A116T-N218S-K256R, A088T-A116T-N218S-N243V-Q271R,         A088T-A116T-N218S-N243V-S248N-K256R,         A088T-A116T-N218S-N243V-S248N-K256R-Q275R,         A088T-A116T-N218S-S248N, A088T-A116T-N218S-S248N-K256R,         A088T-A116T-N243V-S248N-K256R, A088T-A116T-T158S,         A088T-A116T-T158S-N218S-K256R,         A088T-A116T-T158S-N218S-N243V-L257G,         A088T-A116T-T158S-N218S-N243V-S248N-L257G,         A088T-A116T-T158S-N218S-S248N,         A088T-A116T-T158S-N218S-S248N-K256R,         A088T-A116T-T158S-N218S-S248N-K256R-L257G,         A088T-A116T-T158S-N218S-S248N-L257G,         A088T-A116T-T158S-S248N-L257G, A088T-G131H, A088T-G131H,         A088T-G131H-N218S-K237R-K256R-L257G, A088T-G131H-N218S-K256R,         A088T-G131H-N218S-K256R-L257G,         A088T-G131H-N218S-N243V-K256R-L257G,         A088T-G131H-N218S-N243V-L257G, A088T-G131H-N218S-N243V-L257G,         A088T-G131H-N218S-N243V-S248N,         A088T-G131H-N218S-N243V-S248N-K256R,         A088T-G131H-N218S-N243V-S248N-K256R-L257G,         A088T-G131H-N218S-N243V-S248N-K256R-L257G,         A088T-G131H-N218S-S248N, A088T-G131H-N243V,         A088T-G131H-N243V-K256R, A088T-G131H-N243V-K256R-L257G,         A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N,         A088T-G131H-N243V-S248N-K256R, A088T-G131H-S248N,         A088T-G131H-S248N-K256R, A088T-G131H-T158S-N218S-K256R-L257G,         A088T-G131H-T158S-N218S-L257G, A088T-G131H-T158S-N218S-N243V,         A088T-G131H-T158S-N218S-N243V-K256R-L257G,         A088T-G131H-T158S-N218S-N243V-S248N-K256R,         A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-G131H-T158S-N218S-N243V-S248N-L257G,         A088T-G131H-T158S-N218S-S248N-K256R,         A088T-G131H-T158S-N218S-S248N-K256R-L257G,         A088T-G131H-T158S-S248N-K256R-L257G, A088T-L257G,         A088T-N109G-A116T-G131H-N218S-L257G,         A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R,         A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-N218S-N243V-S248N-N269D,         A088T-N109G-A116T-G131H-N218S-N243V-S248N-Q275R,         A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-N243V-S248N,         A088T-N109G-A116T-G131H-T158S,         A088T-N109G-A116T-G131H-T158S-N218S-L257G-I268V,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,         A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G,         A088T-N109G-A116T-G131H-T158S-S248N,         A088T-N109G-A116T-G131H-W241L-S248N-K256R-L257G,         A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R-L257G,         A088T-N109G-A116T-T158S-N218S-K256R-L257G,         A088T-N109G-A116T-T158S-N218S-N243V-S248N-L257G,         A088T-N109G-A116T-T158S-N243V-S248N-K256R,         A088T-N109G-G131H-A138V-T158S-N218S-N243V-S248N-L257G,         A088T-N109G-G131H-N218S-N243V-S248N-L257G,         A088T-N109G-G131H-N218S-S248N-K256R-L257G-Q275R,         A088T-N109G-G131H-N243V-S248N,         A088T-N109G-G131H-N243V-S248N-K256R-L257G,         A088T-N109G-G131H-T158S-L233S-N243V-S248N,         A088T-N109G-G131H-T158S-N218S-N243V-K256R-L257G,         A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-G131H-T158S-N218S-N243V-S248N-L257G,         A088T-N109G-G131H-T158S-N218S-S248N-K256R,         A088T-N109G-G131H-T158S-N243V-S248N-K256R,         A088T-N109G-G131H-T158S-S248N-K256R,         A088T-N109G-G131H-T158S-S248N-K256R-L257G,         A088T-N109G-N218S-K256R-L257G, A088T-N109G-N218S-N243V-K256R,         A088T-N109G-N218S-N243V-S248N,         A088T-N109G-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-N218S-S248N-K256R, A088T-N109G-N243V-K256R,         A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S,         A088T-N109G-T158S-K256R-L257G,         A088T-N109G-T158S-N218S-N243V-K256R,         A088T-N109G-T158S-N218S-N243V-K256R-L257G,         A088T-N109G-T158S-N218S-N243V-S248N-K256R,         A088T-N109G-T158S-N218S-N243V-S248N-L257G,         A088T-N109G-T158S-S248N, A088T-N218S-N243V-S248N,         A088T-N218S-N243V-S248N-K256R, A088T-N218S-N243V-S248N-K256R,         A088T-N218S-S248N, A088T-N218S-S248N-L257G,         A088T-S248N-K256R-L257G, A088T-T158S-K256R,         A088T-T158S-N218S-N243V-K256R, A088T-T158S-N218S-N243V-L257G,         A088T-T158S-N218S-N243V-S248N,         A088T-T158S-N218S-N243V-S248N-K256R,         A088T-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-T158S-N218S-S248N-K256R, A088T-T158S-N218S-S248N-L257G,         A088T-T158S-N218S-S248N-L257G-Q275K, A088T-T158S-N243V,         A088T-T158S-N243V-S248N, A088T-T158S-S248N-K256R-L257G,         A088T-V147I-N218S-N243V-K256R-L257G, A116T-G131H-L257G,         A116T-G131H-N218S-N243V, A116T-G131H-N218S-N243V-K256R-L257G,         A116T-G131H-N218S-N243V-S248N-K256R-L257G,         A116T-G131H-N218S-S248N, A116T-G131H-N218S-S248N-K256R,         A116T-G131H-N243V-S248N-K256R,         A116T-G131H-T158S-N218S-N243V-S248N,         A116T-G131H-T158S-N218S-S248N, A116T-G131H-T158S-N243V-K256R,         A116T-G131H-V130I-N218S-N243V-S248N,         A116T-K141E-N218S-N243V-S248N-K256R-L257G,         A116T-N218S-N243V-S248N, A116T-N218T-N243V-S248N,         A116T-N243V-K256R-L257G, A116T-S248N-K256R,         A116T-T158S-N218S-K256R, A116T-T158S-N218S-K256R-L257G,         A116T-T158S-N218S-N243V-L257G, A116T-T158S-N218S-N243V-S248N,         A116T-T158S-N218S-S248N-L257G, G024S-G053S-N078S-G097A-N101S,         G053S-A088T-N109G-A116T-G131H-T158S-G169S-N218S-S248N-K256R-L257G,         G131H-K141R-T158S-N218S-K256R, G131H-K256R,         G131H-N218S-K256R-L257G, G131H-N218S-N243V-S248N-L257G,         G131H-N218S-S248N-K256R, G131H-N243V-S248N,         G131H-N243V-S248N-L257G, G131H-T158S-N218S,         G131H-T158S-N218S-N240H-N243V-S248N-K256R-L257G,         G131H-T158S-N218S-N243V,         G131H-T158S-N218S-S248N-K256R-L257G-N269S,         G131H-T158S-N243V-L257G, G131H-T158S-N243V-S248N,         G131H-T158S-S248N, K256R, N109G-A116T-G131H-N218S-N243V,         N109G-A116T-G131H-N218S-N243V-L257G,         N109G-A116T-G131H-N218S-S248N-L257G,         N109G-A116T-G131H-N218S-W241R-N243V-K256R,         N109G-A116T-G131H-S248N-L257G,         N109G-A116T-G131H-T158S-N218S-K256R,         N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G,         N109G-A116T-G131H-T158S-N218S-N243V-S248N,         N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G,         N109G-A116T-G131H-T158S-N243V-K256R-L257G,         N109G-A116T-G131H-T158S-N243V-S248N-K256R,         N109G-A116T-G131H-T158S-S248N-K256R-L257G,         N109G-A116T-I234T-N243V-S248N-K256R-L257G,         N109G-A116T-N218S-N243V-S248N, N109G-A116T-N243V-K256R-L257G,         N109G-A116T-T158S-N218S-K237R-N243V-S248N,         N109G-G131H-N218S-N243V-S248N, N109G-G131H-S248N,         N109G-G131H-T158S, N109G-G131H-T158S-L257G,         N109G-G131H-T158S-N218S-N243V-S248N-K256R,         N109G-G131H-T158S-N218S-S248N-K256R,         N109G-G131H-T158S-N218S-S248N-L257G,         N109G-G131H-T158S-N243V-S248N-K256R-L257G,         N109G-G131H-T158S-S248N-K256R, N109G-N218S-K256R-L257G,         N109G-N218S-N243V-S248N-K256R, N109G-N218S-S248N-L257G,         N109G-S248N, N109G-T158S-N218S, N109G-T158S-N218S-N243V,         N109G-T158S-N243V-L257G, N218S-N243V-K256R, N218S-N243V-S248N,         N218S-S248N, N218S-S248N-K256R, N243V-L257G,         S003P-N109G-A116T-G131H-N218S-N243V-S248N,         S003P-N109G-A116T-G131H-T158S-N218S-K256R,         S105H-W106G-I107L-I108S-N109A-G110A-I111S-E112N-W113G-A114P,         S248N-L257G, T158S-N218S-K256R, T158S-N218S-L233S-S248N,         T158S-N218S-L257G, T158S-N218S-N243V-K256R,         T158S-N218S-N243V-S248N, T158S-N218S-N243V-S248N-L257G,         T158S-N218S-S248N-K256R-L257G, T158S-N218S-S248N-L257G,         T158S-N243V-S248N-L257G,         V004L-A088T-G131H-T158S-N218S-S248N-L257G, A133V-S260N,         N061S-S260P, P014T-S037T, S009T-K141F, S009T-K141R, S018F-S162L,         S018L-Y021S, S018P-D120N, S018T-S162P, S018T-Y021N, S018Y-K213R,         S161P-S162L, S161P-S260P, T253A-S260P, A134T-S260G, I115V-N184Y,         N025K-S037P, Q010L-S037P, Q019L-S260N, Q019L-S260P, S037P-T254S,         S161P-T253A, N061S-S260P, Q010L-S037P, S009T-K141F, S018L-Y021S,         S018T-S162P, S018T-Y021N, S018Y-K213R, S037P-T254S, S161P-S260P,         S161P-T253A, T253A-S260P, A133V-S260N, A134T-S260G, I115T-S183T,         I115V-N184Y, N061D-S260I, Q019L-S260N, Q019L-S260P, S183T-S249R,         N61D-Q206R, Y262N-Q275R, K43R-N76S, K170R-D259G, Y6F-S249C,         Q19A-N109S, H17L-Q19A, Q19R-Q185R, S18Y-V203A, S161E-S260T,         S18K-V203I, V4A-T55A, N252S-L257H, S249R-Y262H, N61L-Q206H,         N184Y-Y262N, Q19R-N25D, A74S-P129Q, K27R-D120H, V4A-T55A,         Y21H-N252H, K27R-N269D, A98T-T158A, I79V-Q217H, S9L-N218S,         V4A-Y6F, V203A-Q217R, T22S-T242S, N76P-N212S, S37T-S260P,         T55A-V147A, V4A-Y6F, Y262N-Q275R, G160R-T244A, N25D-Q185R,         G211V-T244A, S9L-N218S, A144H-T244A, Y21H-N252H, A1Y-Q275R,         V198L-D259G, K141I-S248N, S183T-R186K, S161E-Q185H, P129S-K136R,         K43N-S163T, S37G-Q275H, Y6F-S249C, N62Y-T244A, S260P-Q275R,         S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V,         S33T-N61G-S63G-N109G-A128S-N218S-N243V,         S33T-S63G-N109G-A128S-N218S-N243V,         S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,         N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q,         S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V,         S33T-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V,         S33T-N76D-N109G-A128S-N218S-N243V-S248N-K256R,         S33T-N61G-N109G-A128S-N218S-N243V, S33T-A128S-N218S,         A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V,         N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q,         N61P-S63G-N109Q-A128S-G131H-S224A-N243V,         S63G-N109Q-A128S-G131H-S224A-N243V, N109G-A114S-A128S,         N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A,         N109G-A114S-A128S-S224A-N243V,         A88T-N109G-A116T-A128S-S224A-N243V,         N61G-A88T-N109G-A116T-A128S-S224A-N243V,         N109G-A114S-A128S-N243V, N109G-A128S-S224A-N243V,         N109G-A128S-S224A, N109G-A128S-S183L-S224A,         N61G-N109G-A128S-S224A, N109G-A128S-S183L, S33T-N76D,         N109S-A128S-S224A, N101Q-N109Q-A128S-P129S-S130T-S224A-N243V,         S63G-N109Q-A128S-S224A-N243V, N109M-A128S-S224A-N243V,         S63G-N109G, N109G-K256R, S63G-N76D,         S33T-N109G-A128S-G169A-N218S-N243V,         S33T-N109G-A128S-N218S-S224A-N243V, N062L, N062L-S063G,         N062S-S063G-Q217L, N062S-S063L-Q217L, N062S-S063N, N062S-S063R,         S063G, S063G-Q217L, S063G-Q217L-M222S, S063L-Q217L, S063N,         S063N-Q217L, D099N-K141Y-K213Q, D099N-K141Y-K256Q,         K043T-K141Y-E156Q, N062L-Q217E, N062L-Q217L, N062L-S063G-Q217E,         N062L-S063L, N062L-S063N-Q217L, N062S-Q217L, N062S-S063G,         N062S-S063L, N062S-S063N-Q217L, N062S-S063R-Q217E, S063G-Q217E,         S063N-Q217E, S063R, S063R-Q217E and S063R-Q217L; -   e) a protease, said protease being selected from the group     consisting of     -   (i) a variant of a parent protease, said parent protease having         at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98%         or 99% or even 100% identity to SEQ ID NO:4, said variant         comprising a group of mutations selected from the following         groups of mutations: Q059V-S078N-I108V-V147Q-G211A-N252Q,         S078N-I108V-V147Q-G211A, S078N-S087E-M124I-S224A,         S078N-I108V-V147Q-N252Q, S078N-I108V-V147Q-G211A-N252Q, and         Q059V-S078N-I108V-V147Q-G211A-N252Q;     -   (ii) a variant of a parent protease, said parent protease,         having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97%         or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant         comprising a group of mutations selected from the following         groups of mutations: G097A-M124V-Y167A-Y217Q, V068A-Y167A-Y217Q,         G097A-I111V-M124V-Y167A, I111V-M124V-Y167A-Y217Q,         V068A-I111V-Y167A-Y217Q, G097A-I111V-M124V-Y167A-Y217Q,         P052L-V068A-I111V, G097A-N123A-Y217Q,         N061P-N062Q-G097A-G100D-Y217Q, N061P-S101N-G102A-G128S-Y217Q,         Y217Q, N061P-G102A-G128S-Y217Q, N061P-S101N-G102A-G128S-Y217Q,         S078N-G097A-I111V-N123Q-Y217Q, G102A-N123Q-Y217Q,         N061P-G102A-G128S-Y217Q, S078N-G097A-I111V-N123Q-Y217Q, and         G102A-N123Q-Y217Q; and     -   (iii) a variant of a parent protease, said parent protease         having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97%         or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant         comprising three, four, five or six of the following mutations:         X24G/R, X53G, X78N, X101N, X128A/S and X217L/Q, said variant         optionally comprising at least one group of mutations selected         from the following groups of mutations: A001D, A001H, A001N,         A015C, A048C, A048E, A085T, A133R, A137R, A142C, A144D, A144R,         A152S, A153G, A187P, A187Q, A187T, A187V, A216R, A230S, A272R,         A273H, A273T, A274H, D036N, D036S, D181H, D181T, D259N, D259P,         D259S, E156G, E156H, E156L, E156Q, E156V, E251C, F189S, F189T,         F189W, F189Y, F261E, G020C, G024D, G053M, G053R, G097R, G131D,         G131R, G157N, G160R, G160V, G166L, G166W, G211E, G215D, G258A,         G258D, G258P, I011T, I031C, I079E, I079R, I175L, I205C, K012H,         K012N, K027A, K027N, K027S, K043A, K043D, K043E, K043G, K043L,         K043M, K043P, K043V, K043W, K043Y, K136H, K141H, K141L, K141M,         K141N, K141Q, K141T, K141V, K170G, K170S, K237T, K237V, K256D,         K256S, K256T, K256V, K265N, K265S, L042F, L042M, L082E, L209A,         L209E, L209G, L209R, L233G, L235V, L257D, L257E, L257P, L257R,         L257W, L267E, M050L, N056D, N056S, N061C, N061D, N062A, N062H,         N062L, N062V, N062Y, N076D, N076L, N076M, N078D, N078F, N101D,         N101R, N118A, N212C, N212E, N218C, N218D, N218E, N252D, N252E,         P014F, P014K, P057A, P057W, P172R, P194E, P201T, P210E, Q059C,         Q059D, Q059R, Q185E, Q206D, Q217A, Q217K, Q217R, Q245A, Q245D,         Q245E, Q245H, Q245R, Q271E, Q271F, Q271R, Q271W, Q275G,         Q275IR186I, R186L, R186V, R186W, S003E, S009C, S009E, S018C,         S037D, S037E, S037H, S037R, S037Y, S038D, S038P, S038R, S038Y,         S063L, S087D, S087R, S089C, S089E, S130C, S130R, S145D, S159C,         S159P, S161C, S173T, S182C, S188E, S188F, S188K, S188L, S188R,         S188W, S190A, S190G, S190T, S204R, S236D, S236E, S248C, S248E,         S260C, S260E, S260R, T022P, T055C, T055W, T071A, T158D, T158E,         T158P, T158R, T158Y, T164R, T242D, T242G, T255C, V004E, V004T,         V045C, V045D, V045R, V045T, V051H, V081R, V143C, V143E, V143G,         V192G, V203C, V203D, V203E, V203M, V203R, V270G, W241L, Y214H,         Y214Q, A001E, A133E, A187L, A187N, A216C, A216H, A273Q, D099H,         D259H, E156C, E195G, F189H, G131C, G146A, G166V, G215C, G215E,         I107L, K012A, K012S, K012T, K043C, K170C, K256C, K256E, K265G,         K265Y, L233EM222F, M222S, N062Q, N076E, N078E, N184P, N218R,         P005V, P014D, Q002K, Q002L, Q002R, Q010W, Q271C, R186H, S049C,         S063C, S063D, S105T, S188C, S190C, S204E, T055R, T164G, V004D,         V044T, V045I, V165C, V180S, Y006C, Y006D, Y006E, Y104T, A001C,         A187C, A230G, A273D, A273P, D036Q, F189G, F189L, F189R, G157T,         G178A, I031F, I111M, K012F, K012L, K027T, K043R, K136G, K141G,         K170Q, M222A, M222L, N062R, N117G, N269C, P005W, P129V, P239A,         P239H, P239T, Q059W, Q217G, Q275A, R186A, S191G, T164A, T220A,         A001P, A187F, A187W, A273R, D041C, D060G, D197T, F189A, G046D,         G157P, K012C, K012E, K012W, L042C, M222T, N062C, P239G, P239N,         Q217C, R186M, S049T, S089P, S125A, S173V, V044A, A001E,         A001E-A128S, A001E-G024E, A001E-G131H, A001E-G169A, A001E-L257G,         A001E-218S, A001E-Q103H, A001E-S063G, A001E-S248N, A001E-S249A,         G024E-N076D, K043Y-N076D, K043Y-Q206D, N076D-A116T, N076D-G169A,         N076D-Q206D, Q103H-Q206D, S033T-G169A,         S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P,         S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, A001E-K043Y,         A001E-N076D, A001E-N076D-N109G-A128S, A001E-Q206D, G024E-Q206D,         A001E-A128S-G131H-N243V, A001E-G131H-G169A-N243V,         A001E-S033T-N109G-N218S, A116T-G169A, A116T-Q206D, G169A-S249A,         K043Y-G169A, N109G-G169A, P040E-N109G-I28S-G131H, Q206D-L257G,         S033T-A128S-G131H-N243P, S033T-A128S-G131H-N243V,         S033T-P040E-Q103H-N109G, S033T-Q103H-A128S-G131H,         S063G-N109G-A128S-G131H, S063G-Q206D, T158S-Q206D,         A001E-N076D-N109G-A128S,         A015S-A088T-N109G-G131H-T158S-N218S-S248N,         A088T-A098S-G131H-S248N-K256R-L257G,         A088T-A116T-G131H-N218S-N243V-K256R-L257G,         A088T-A116T-G131H-T158S-L257G,         A088T-A116T-G131H-T158S-N218S-L257G,         A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G,         A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G,         A088T-A116T-N218S-L257G, A088T-A116T-T158S-K256R,         A088T-A116T-T158S-S248N-K256R-L257G,         A088T-G131H-K141E-N218S-N243V-S248N-L257G, A088T-G131H-K256R,         A088T-G131H-N218S-K256R, A088T-G131H-N218S-N243V-S248N-K256R,         A088T-G131H-N218S-N243V-S248N-L257G,         A088T-G131H-N218S-S248N-K256R, A088T-G131H-T158S-S248N-K256R,         A088T-G131H-T158S-S248N-K256R-L257G,         A088T-I107T-N109G-G131H-N218S-S248N-K256R,         A088T-N109G-A116T-G131H-D140G-T158S-N218S-N243V-K256R,         A088T-N109G-A116T-G131H-N218S-N243V-K256R,         A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R,         A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G,         A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-I268V,         A088T-N109G-A116T-G131H-V149A-N218S-S248N-K256R-L257G,         A088T-N109G-A116T-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-T158S-K256R-L257G,         A088T-N109G-A116T-T158S-N218S-N243V-L257G,         A088T-N109G-D140G-N243V,         A088T-N109G-G131H-D140G-T158S-N243V-S248N-K256R,         A088T-N109G-G131H-K141E-T158S-N218S-K256R,         A088T-N109G-G131H-N218S-S248N,         A088T-N109G-G131H-N218S-S248N-K256R-Q271R,         A088T-N109G-G131H-N218S-S248N-L257G,         A088T-N109G-G131H-T158S-K256R,         A088T-N109G-G131H-T158S-N218S-S248N-K256R,         A088T-N109G-G131H-V149L-T158S-K256R-L257G,         A088T-N109G-T158S-N218S,         A088T-N109G-T158S-N218S-K256R-L257G-Q271K,         A088T-N109G-T158S-N218S-L257G, A088T-N109G-T158S-S248N-K256R,         A088T-N218S-S248N-L257G-Q27R, A088T-T158S-N218S-K256R-L257G,         A088T-T158S-N218S-N243V-K256R,         A088T-Y104H-A116T-G131H-N218S-N243V,         A116T-G131H-K141E-N218S-N243V-S248N-L257G,         A116T-G131H-N218S-N243V-S248N-K256R,         A116T-G131H-T158S-N218S-S248N-L257G-N269D,         A116T-G131H-T158S-N218S-S248N-Q271R,         A116T-G131H-T158S-N243V-S248N,         A116T-G157E-T158S-N243V-S248N-K256R, A116T-T158S-N218S,         G131H-N218S-L257G, G131H-N218S-S248N,         G131H-T158S-N218S-N243V-S248N-K256R-L257G,         G131H-T158S-N218S-N243V-S248N-L257G,         G131H-T158S-N218S-S248N-I268V, I107T-N109G-G131H-N218S-L257G,         L090I-N109G-T158S-N243V, L257G,         N109G-A116T-G131H-T158S-N218S-K256R-L257G-Q271R,         N109G-A116T-N218S-W241R-N243V-S248N-K256R-L257G,         N109G-G131H-K141E-L257G, N109G-G131H-N218S-N243V,         N109G-T158S-N218S-N243V-L257G, N109G-T158S-N218S-S248N-K256R,         N109G-T158S-N243V-S248N-K256R-L257G, N218S-S248N-K256R-L257G,         S003P-N109G-G131H-T158S-L257G, S003P-S248N-L257G,         T158S-S248N-K256R-L257G,         V004A-A088T-G131H-N218S-N243V-S248N-L257G,         Y006H-N218S-N243V-S248N, Y104H-N109G-G131H-N243V-S248N,         A088T-A116T-T158S-N218S-N243V-S248N-K256R,         A088T-A116T-T158S-N243V,         A088T-G131H-T158S-N218S-I234T-S248N-L257G,         A088T-G131H-T158S-N218S-N243V-S248N-K256R,         A088T-G131H-V149L-T158S-N243V-S248N-K256R-L257G,         A088T-I107T-N109G-G131H-N218S-A223G-S248N-K256R,         A088T-K213N-N243V-S248N-K256R, A088T-K256R-L257G,         A088T-N109G-A116T-G131H-A232S-N243V-K256R,         A088T-N109G-A116T-G131H-D140G-S248N-L257G,         A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,         A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G,         A088T-N109G-A116T-M124I-G131H-T158S-N218S-S248N-L257G,         A088T-N109G-A116T-V148A-N218S-N243V,         A088T-N109G-G131H-N218S-N243V-S248N,         A088T-N109G-N218S-S248N-T255K-K256R-L257G,         A088T-T158S-N218S-L257G, A088T-T158S-N218S-Q245K-S248N-K256R,         A088T-T158S-N218S-S248N-K256R, A116T-G131H-N218S-N243V-K256R,         A116T-G131H-N218S-W241R-N243V-S248N-K256R-L257G,         A116T-G131H-T158S-N218S-L257G,         A116T-G131H-V150A-T158S-N243V-S248N-K256R-L257G,         I107T-G131H-T158S-N243V-S248N-K256R-L257G,         N109G-A116T-K141E-T158S-N218S-N243V-L257G,         N109G-A116T-T158S-N218S-N243V-S248N, T158S-N243V-S248N-K256R,         T158S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-G146C,         A088T-A116T-N218S, A088T-A116T-T158S-N243V-K256R-L257G,         A088T-A138E-N218S-N243V-K256R,         A088T-N109G-A116T-G131H-T158S-N218S-N243F-S248N,         A088T-T158S-V203I-N218S-K256R-L257G,         A116T-D140G-T158S-N218S-N243V-S248N,         A088T-A116T-T158S-K256R-L257G,         A088T-A116T-T158S-N218S-N243V-S248N-E251K-K256R-L257G,         A088T-I108T-N109G-G131H-T158S-N218S-S248N-K256R-L257G,         A088T-N109G-A116T-G131H-K141E-N218S,         A088T-N109G-W241R-S248N-K256R, G065D-A088T-G131H-N243V-S248N,         A045S-S236G, G024A-S037W, I031V-S038W, N061D-S260I, Q010R-S037T,         I115T-S183T, N025K-P129K, N025K-P129R, A045S-S236Y, S162L-D181H,         I031V-S038W, N025K-S037P, N025K-P129R, Y21H-D259G, A133V-D259N,         I79V-Q217H, S18K-V203I, T158A-D259P, N61K-N252K, K43N-Q217R,         Q206R-S260P, V198L-D259G, S161E-S260T, G160A-D259G, A1Y-Q275R,         A200T-H226L, Q217R-T244A, S260P-Q275R, T158I-D259N, L75I-N76D,         S161E-Q185H, Y21H-S37E, S249R-Q275R, N76T-N212D, S260P-Q275L,         G131S-K265N, V4A-S249N, N25D-Q185R, K43R-N76S, S183D-Q206R,         Q10R-Q275K, K43N-S163T, N25D-V26A, S260P-Q275L, K141I-S248N,         L16Q-Q217H, K27R-N269T, P210S-N212D, N118G-V121A, G215D-D259V,         N62Y-G97D, V4E-S260P, K27E-Y91F, Y6D-T55A, N77D-N252T,         N25K-H238R, V44A-Q206H, S37P-S260F, V44A-Q206R,         I31L-S33T-S63G-N109G-A128S-G169A-N218S-N243V,         A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q,         S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V,         S33T-S63G-N109G-A128S-G169A-N218S-N243V,         S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q,         S33T-N76D-A128S-N218S, N76D-N109G-A128S-S224A, D099N,         K141Y-E156Q, N062L-S063L-Q217L, N062L-S063N, N062L-S063N-Q217E,         N062L-S063R, N062L-S063R-Q217L, N062S-Q217E, N062S-S063G-Q217E,         N062S-S063G-Q217R, N062S-S063N-Q217R, S063G-S125A, D060G-Q217L,         D120N-K141Y-K213Q, K043T-D099N-D120N-K141Y,         K043T-D099N-K141Y-K256Q, K043T-K237A, N062L-S063G-Q217R,         N062L-S063G-S125A, N062L-S063L-Q217E, N062L-S063N-S125A-Q217L,         N062S-Q217R, N062S-S063L-Q217E, N062S-S063R-Q217L, S063G-M222S,         S063G-Q217R, K141Y-D197N, N062L-Q217R, N062L-S063G-Q217L-M222S,         N062L-S063L-Q217R, N062L-S063N-Q217R, N062S-Q217G,         N062S-S063G-Q217G, N062S-S063G-Q217L-M222L,         N062S-S063G-S125A-Q217L, N062S-S063N-Q217E, Q217G,         S033G-N062S-S063G, S063G-Q217G, S063G-Q217L-M222L,         S063G-S125A-Q217R, S063L-Q217R, S063N-M222S, S063N-Q217R,         S063N-S125A-Q217L, S063R-Q217R, S063R-S125A-Q217L,         D099N-E156Q-K256Q, E156Q, K012T-D099N-K213Q, K012T-K256Q,         K043T-D099N-K141Y-K213Q, K043T-E156Q, K141Y-K213Q, N062L-Q217G,         N062L-Q217L-M222L, N062L-Q217L-M222S, N062L-S063G-M222S,         N062L-S063G-Q217L-M222L, N062L-S063G-Q217R-M222S,         N062L-S063N-Q217L-M222S, N062L-S063N-S125A, N062L-S063R-S125A,         N062L-S125A, N062S-S063G-M222S, N062S-S063G-Q217G-M222S,         N062S-S063G-S125A, N062S-S063N-Q217L-M222L,         N062S-S063N-S125A-Q217L, N062S-S063R-Q217G,         N062S-S063R-Q217L-M222S, Q217G-M222S, Q217L-M222S, Q217R,         S033G-S063G-Q217R, S063G-Q217E-M222S, S063G-S125A-Q217G,         S063L-Q217E, S063N-Q217G, S063N-Q217G-M222S, S063N-Q217L-M222S,         S063R-Q217L-M222S and S063R-S125A. -   f) a variant of a parent protease, the variant comprising an amino     acid sequence comprising from three to six amino acid substitutions     selected from the group consisting of X024G/R, X053G, X078N, X101N,     X128A/S, and X217L/Q, wherein the variant has proteolytic activity; -   g) a variant of a protease, the variant having proteolytic activity     and comprising an amino acid sequence which comprises from two to     seven alterations at the amino acid positions corresponding to amino     acid positions of SEQ ID NO:2 selected from the group consisting of     positions 24, 53, 78, 97, 101, 128, and 217, wherein each alteration     is independently     -   (i) an insertion of one or more amino acid residues upstream or         downstream of the amino acid residue which occupies the         position,     -   (ii) a deletion of the amino acid residue which occupies the         position, or     -   (iii) a substitution of the amino acid residue which occupies         the position with a different amino acid residue, -   h) a variant of a parent protease, wherein: the variant comprises an     amino acid sequence having no more than 15 alterations relative to     the parent protease, wherein the alterations are independently     selected from an insertion, a deletion, or a substitution, and the     alterations include a substitution of glycine at positions 24 and     53, a substitution of asparagine at positions 78 and 101, a     substitution of alanine or serine at position 128, and a     substitution of glutamine at position 217; the parent protease has     at least 90% sequence identity to SEQ ID NO:2; and the variant has     increased proteolytic activity relative to the parent protease -   i) a variant of a parent protease, said parent protease at least     90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to SEQ     ID NO:2, said variant having an amino acid sequence having at least     90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identity to the sequence     of SEQ IDNO:2; said variant comprising an a substitution of glycine     at positions 24 and 53, a substitution of asparagine at positions 78     and 101, a substitution of alanine or serine at position 128, and a     substitution of glutamine at position 217, and said variant having     increased proteolytic activity relative to the parent protease; -   j) mixtures thereof.     and one or more adjunct ingredients, said composition being a fabric     and home care product is disclosed.

In one aspect of said composition, said cold water protease has either a performance index greater than 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2 from at least 1 to about 10, from at least 1 to about 8 or even from at least 1 to about 5 on BMI at pH 8 and 60° F. when compared to an enzyme having SEQ ID NO:4, as defined in Test Method 2 or Test Method 3 or said cold water protease has a performance index at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2 from 1.0 to about 10, from 1.0 to about 8 or even from 1.0 to about 5 on BMI at pH 8 and 60° F. when compared to an enzyme having SEQ ID NO:6, as defined in Test Method 2 or Test Method Sand said cold water protease is selected from the group consisting of:

-   a) a variant of a parent protease having SEQ ID NO:4, said variant     comprising a group of mutations selected from the following groups     of mutations: P210S, P210S-N218A, S063T-S078N-S101A-S183T-T244N,     N061A-S078N-S224A, S053G-S078N-P129T-Q185T, S063T-S078N-S101A,     S078N-P129T, S063T-S078N-S101A-S183T and S063T-S078N-S101A-T2441; -   b) a variant of a parent protease having SEQ ID NO:2, said variant     comprising a group of mutations selected from the following groups     of mutations: G097A-I111V-M124V-Y217Q, G097A-I111V-Y167A-Y217Q,     S024G-N025G-N061P-G097A-S101N-G128S-Y217Q,     S024G-N025G-S053G-N061P-G097A-S101N-G128A-V203Y-Y217Q,     S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128S-V203Y-Y217Q,     V068A-A092G-Y217Q, N061P-G097A-S101N-G128A-P201S-Y217Q,     S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q,     S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q,     S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q,     S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,     N025G-G097A-S101N-G128A-Y217Q,     N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,     N025G-S053G-N061P-S078N-G128A-Y217Q,     N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,     N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,     N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,     N025G-S078N-G097A-S101N-G128A-Y217Q,     N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,     N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q,     S024G-N025G-N061P-G097A-G128A-Y217Q,     S024G-N025G-N061P-G097A-S101N-G128A-Y217Q,     S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q,     S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q,     S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q,     S024G-N025G-S053G-T055P-N061P-G128A-Y217Q,     S024G-N025G-T055P-G097A-G128A-Y217Q,     S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,     S024G-N061P-S078N-G097A-S101N-G128A-Y217Q,     S024G-S053G-N061P-G097A-G128A-Y217Q,     S024G-S053G-N061P-S078N-G097A-G128A-Y217Q,     S024G-S053G-T055P-G097A-S101N-G128A-Y217Q,     S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,     S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,     S024G-S053G-T055P-N061P-S101N-G128A-Y217Q,     S024G-T055P-N061P-G097A-G128A-Y217Q, S053G-G097A-S101N-G128A-Y217Q,     S053G-N061P-G097A-S101N-G128A-Y217Q-S249N,     S053G-N061P-S078N-G097A-G128A-Y217Q,     S053G-S078N-G097A-S101N-G128A-Y217Q,     S053G-T055P-G097A-S101N-G128A-Y217Q,     S053G-T055P-N061P-S101N-G128A-Y217Q,     S053G-T055P-S078N-G097A-S101N-G128A-Y217Q,     T055P-G097A-S101N-G128A-Y217Q,     T055P-N061P-S078N-G097A-S101N-G128A-Y217Q,     S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q,     S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q,     S024G-T055P-N061P-S078N-S101N-G128A-Y217Q,     T055P-N061P-S078N-G128A-Y217Q and S24G-S53G-S78N-S101N-G128A-Y217Q; -   c) a variant of a parent protease having SEQ ID NO:6, said variant     comprising a group of mutations selected from the following groups     of mutations: A116V, G160S, I111L, I115V, N109S, N117M, P005G,     Q059V, T164S, Y262M, A015Q, A015S, A098E, A098N, A098S, A098T,     A098V, A098Y, A114S, A114T, A116G, A116L, A116S, A116T, A116W,     A133G, A133H, A133T, A133V, A137G, A137I, A137L, A137S, A137T,     A138S, A216E, A216F, A216V, D099S, D181E, F261A, F261Q, G024F,     G024I, G024Q, G024Y, G097S, G160T, G211L, G211V, H017F, H017W,     H039V, H226A, I031V, I111V, I268V, K170R, K265R, L016Q, L016T,     L135M, L209T, L209V, L233M, L257T, L257V, L267A, L267V, N025A,     N025I, N025 Q, N025 R, N025 T, N025 V, N101I, N101Q, N101S, N109A,     N109G, N109H, N109L, N109M, N109Q, N109T, N117Q, N184A, N184L,     N184T, N184W, N212G, N212L, N212V, N243P, N252G, N252M, P005T,     P014S, P040G, P040L, P040Q, P129A, P129S, P172G, P172S, P194Q,     P210A, P210S, Q185F, Q185G, Q185I, Q185M, Q185N, Q185S, Q275H,     R186K, S009A, S009G, S009H, S009M, S018T, S130T, S132N, S145K,     S159T, S161I, S161K, S161N, S161T, S162I, S162M, S162Y, S163G,     S182F, S182G, S182V, S182W, S183F, S183L, S183M, S183T, S183V,     S183W, S224A, S236T, S249V, T022A, T022G, T022Q, T022V, T208V,     T242S, T253N, T253S, T254A, T254S, T255L, T255S, T255V, V004A,     V004P, V004W, V084C, V139C, V165M, V203F, Y021K, Y021N, Y021T,     Y021V, Y167F, Y171F, Y214F, Y262F, Y262T, A088T-L257G, A116T-A128S,     N061S-N109G-A128S-N243V-S260P, S009T-N109G-A128S-K141R-N243V,     S009T-S018T-Y021N-N109G-A128S-K141R, S162G-K256R, A088T-N243V,     G024E-A116T, K043Y, N076D-A116T, N218S-S248N, S033T-N243V,     S033T-S063G, S248N-L257G, A001E-S249A, A088T-A116T, A088T-A128S,     A088T-G131H, A088T-N109G, A088T-S248N, A088T-S249A, A116T-N243V,     A116T-T158S, A128S, A128S-K256R, A128S-L257G, A128S-N243V,     A128S-S248N, A128S-T158S, G024E-A088T, G024E-A128S, G024E-G131H,     G024E-K256R, G024E-L257G, G024E-N218S, G024E-N243V, G024E-S162G,     G024E-S249A, G024E-T158S, G131H, G131H-K256R, G131H-S249A,     K043Y-A088T, K043Y-A116T, K256R, N076D-K256R, N109G, N109G-A116T,     N109G-A128S, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A,     N109G-G131H, N109G-K256R, N109G-L257G, N109G-N218S, N109G-N243V,     N109G-S248N, N218S-L257G, N243V, N243V-K256R, N243V-L257G,     N243V-S248N, N243V-S249A, Q103H-A128S, Q103H-G131H, Q103H-K256R,     Q103H-L257G, Q103H-N243V, Q103H-S248N, Q103H-S249A, Q103H-T158S,     Q206D-N243V, S033T-A128S, S033T-K256R, S033T-N076D, S033T-N218S,     S033T-S248N, S033T-T158S, S063G-A128S, S063G-K256R, S063G-N243V,     S063G-S162G, S063G-T158S, S248N-K256R, S249A, T158S-N243V,     T158S-S249A, A088T-A116T-N243V-K256R-L257G, A088T-A116T-N243V-L257G,     A088T-T158S-N218S-K256R, A088T-T158S-N218S-N243V-L257G,     A088T-A116T-T158S-N218S-N243V-K256R-L257G,     A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G,     A088T-N109G-A116T-T158S-S248N-K256R-L257G, A088T-N109G-T158S-L257G,     A114S-A116T-N218S-N243V-S248N-K256R-L257G, A116T-T158S-K256R,     A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-T158S,     A088T-N109G-A116T-G131H-L257G,     A088T-N109G-A116T-T158S-N243V-S248N-L257G, A088T-N109G-N243V-L257G,     A088T-N109G-N243V-S248N, A088T-N109G-T158S-N243V-L257G,     A088T-N109G-T158S-N243V-S248N-L257G, A116T-T158S-S248N-L257G,     Y006H-A116T-G131H-S248N, A088T-A116T-G131H-T158S-N218S-N243V,     A088T-A116T-G131H-T158S-N243V,     A088T-A116T-G131H-T158S-N243V-K256R-L257G,     A088T-A116T-N218S-N243V-K256R-L257G, A088T-A116T-S248N-K256R-L257G,     A088T-A116T-T158S-N218S-N243V, A088T-A116T-T158S-N243V-K256R-L257G,     A088T-A116T-T158S-N243V-S248N-L257G,     A088T-G131H-N243V-S248N-K256R-L257G, A088T-N109G-A116T-T158S-L257G,     A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G,     A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R,     A088T-N109G-A116T-T158S-S248N-L257G,     A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G, A088T-N109G-K256R,     A088T-N109G-N243V-S248N-L257G, A088T-N109G-T158S-K256R     A088T-N109G-T158S-N243V, A088T-T158S-N243V-K256R-L257G,     A116T-N218S-N243V-L257G-N269S, A116T-T158S-K256R-L257G,     N109G-A116T-K256R-L257G, N109G-A116T-N243V,     N109G-A116T-T158S-N243V-K256R-L257G, N109G-G131H-L257G,     N109G-G131H-S248N-K256R-L257G, N109G-G131H-T158S-K256R-L257G,     S003P-A116T-T158S-S248N-K256R, T158S-S248N-K256R,     A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-S248N-K256R-L257G,     A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G,     A088T-A116T-S248N-L257G, A088T-A116T-T158S-N218S,     A088T-A116T-T158S-N218S-K256R-L257G, A088T-A116T-T158S-N218S-L257G,     A088T-G131H-N243V-L257G, A088T-G131H-T158S-S248N-L257G,     A088T-N109G-A116T, A088T-N109G-A116T-G131H-N218S,     A088T-N109G-A116T-G131H-N218S-S248N-L257G,     A088T-N109G-A116T-G131H-N243V-S248N-K256R-L257G,     A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,     A088T-N109G-A116T-N218S-N243V-K256R,     A088T-N109G-A116T-N218S-N243V-L257G,     A088T-N109G-A116T-N243V-S248N-K256R,     A088T-N109G-A116T-N243V-S248N-K256R-L257G,     A088T-N109G-A116T-T158S-N243V-L257G,     A088T-N109G-G131H-T158S-N243V-S248N-K256R,     A088T-N109G-G131H-T158S-W241R-S248N-K256R, A088T-N109G-K256R-L257G,     A088T-N109G-L257G, A088T-N109G-N243V, A088T-N109G-N243V-K256R,     A088T-N109G-N243V-K256R-L257G, A088T-N109G-S248N-K256R,     A088T-N109G-T158S-N218S-K256R-L257G,     A088T-N109G-T158S-N218S-N243V-S248N-K256R,     A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-K256R-L257G,     A088T-N109G-T158S-N243V-S248N-A274D, A088T-N109G-T158S-S248N-L257G,     A088T-T158S-K256R, A088T-T158S-N218S-N243V-K256R-L257G,     A088T-T158S-N243V-L257G, A116T-G131H-N218S-N243V-S248N,     A116T-G131H-S248N-L257G, A116T-S248N-K256R-L257G,     A116T-T158S-N218S-N243V-K256R, A116T-T158S-N218S-S248N-L257G-Q271R,     A116T-T158S-N243V-K256R-L257G, A116T-T158S-N243V-S248N-L257G,     G131H-S248N, G131H-T158S-I234T-N243V-K256R,     G131H-W241L-N243V-S248N-K256R,     N109G-A116T-G131H-A137V-T158S-S248N-K256R-L257G,     N109G-A116T-G131H-A151S-N218S-K256R-L257G,     N109G-A116T-G131H-T158S-N218S-N243V-K256R,     N109G-A116T-G131H-T158S-N218S-S248N,     N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-S248N,     N109G-A116T-T158S-L257G, N109G-A116T-T158S-N218S-W241R-N243V,     N109G-A116T-T158S-N243V-S248N-L257G,     N109G-A116T-T158S-S248N-K256R-L257G, N109G-A116T-T158S-S248N-L257G,     N109G-G131H-N218S-L257G, N109G-G131H-N218S-S248N-K256R-L257G,     N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T, N109G-N243V-L257G,     N109G-T158S-N218S-K256R-L257G, N109G-T158S-N218S-L257G,     N109G-T158S-S248N-K256R,     P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E,     S003F-A088T-N109G-A116T-T158S-N243V-K256R-L257G,     V004A-A088T-A116T-T158S-N218S,     V004A-N109G-A116T-G131H-S248N-K256R-L257G,     V004L-A116T-N218S-N243V-S248N-L257G, Y006H-N109G-N218S-N243V-S248N,     A001T-A116T-T158S-N243V-L257G, A088T-A116T-G131H-L257G,     A088T-A116T-G131H-N218S-L257G,     A088T-A116T-G131H-N218S-S248N-K256R-L257G,     A088T-A116T-G131H-N218S-S248N-L257G,     A088T-A116T-G131H-N243V-K256R-L257G, A088T-A116T-G131H-N243V-L257G,     A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-T158S-K256R-L257G,     A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S,     A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T,     A088T-A116T-G131H-T158S-N218S-S248N-K256R,     A088T-A116T-G131H-T158S-N218S-S248N-L257G,     A088T-A116T-G131H-T158S-N243V-S248N-K256R,     A088T-A116T-G131H-T158S-S248N, A088T-A116T-K256R,     A088T-A116T-K256R-L257G, A088T-A116T-N218S-N243V-L257G,     A088T-A116T-N243V-K256R, A088T-A116T-N243V-S248N-K256R-L257G,     A088T-A116T-S248N-K256R, A088T-A116T-T158S-K256R,     A088T-A116T-T158S-N218S-N243V-K256R,     A088T-A116T-T158S-N218S-N243V-K256R-N269S,     A088T-A116T-T158S-N218S-N243V-S248N,     A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G,     A088T-A116T-T158S-N243V-K256R, A088T-A116T-T158S-N243V-L257G,     A088T-A116T-T158S-N243V-S248N-K256R,     A088T-A116T-T158S-N243V-S248N-K256R-L257G,     A088T-A116T-T158S-S248N-K256R, A088T-A116T-V143A-N218S-S248N-K256R,     A088T-A116T-V147I-T158S-N218S-N243V-L257G, A088T-G131H-K256R-L257G,     A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-S248N-L257G,     A088T-G131H-S248N-K256R-L257G, A088T-G131H-T158S-L257G,     A088T-G131H-T158S-N218S-K256R,     A088T-G131H-T158S-N218S-N243V-K256R-L257G,     A088T-G131H-T158S-N218S-N243V-L257G, A088T-G131H-T158S-N218S-S248N,     A088T-G131H-T158S-N243V, A088T-G131H-T158S-N243V-S248N,     A088T-G131H-T158S-N243V-S248N-K256R,     A088T-G131H-T158S-N243V-S248N-L257G,     A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N,     A088T-N109G-A116T-G131H-N218S-L257G,     A088T-N109G-A116T-G131H-N218S-N243V,     A088T-N109G-A116T-G131H-N218S-N243V-K256R-L257G,     A088T-N109G-A116T-G131H-N218S-N243V-L257G,     A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G,     A088T-N109G-A116T-G131H-N243V, A088T-N109G-A116T-G131H-N243V-L257G,     A088T-N109G-A116T-G131H-N243V-S248N-L257G,     A088T-N109G-A116T-G131H-S248N, A088T-N109G-A116T-G131H-S248N-K256R,     A088T-N109G-A116T-G131H-S248N-L257G,     A088T-N109G-A116T-G131H-T158S-L257G,     A088T-N109G-A116T-G131H-T158S-N218S,     A088T-N109G-A116T-G131H-T158S-N218S-S248N-K256R,     A088T-N109G-A116T-G131H-T158S-N218T-N243V,     A088T-N109G-A116T-G131H-T158S-N243V-K256R,     A088T-N109G-A116T-G131H-T158S-N243V-K256R-L257G,     A088T-N109G-A116T-G131H-T158S-N243V-S248N,     A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R,     A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G,     A088T-N109G-A116T-G131H-T158S-S248N-L257G, A088T-N109G-A116T-N218S,     A088T-N109G-A116T-N218S-L257G, A088T-N109G-A116T-N218S-N243V,     A088T-N109G-A116T-N218S-N243V-S248N-L257G,     A088T-N109G-A116T-N218S-S248N-K256R, A088T-N109G-A116T-N218T-K256R,     A088T-N109G-A116T-N218T-K256R-L257G, A088T-N109G-A116T-N243V,     A088T-N109G-A116T-N243V-K256R-L257G,     A088T-N109G-A116T-N243V-K256R-L257G-N269D,     A088T-N109G-A116T-S248N-K256R, A088T-N109G-A116T-T158S,     A088T-N109G-A116T-T158S-N218S-L257G,     A088T-N109G-A116T-T158S-N218S-N243V,     A088T-N109G-A116T-T158S-N218S-N243V-K256R,     A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G,     A088T-N109G-A116T-T158S-N218S-N243V-L257G,     A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-A116T-T158S-N243V,     A088T-N109G-A116T-T158S-N243V-K256R,     A088T-N109G-A116T-T158S-N243V-K256R-L257G, A088T-N109G-G131H-L257G,     A088T-N109G-G131H-N218S-K256R-L257G,     A088T-N109G-G131H-N218S-N243V-K256R,     A088T-N109G-G131H-N218S-N243V-L257G,     A088T-N109G-G131H-N218S-N243V-S248N-K256R-L257G,     A088T-N109G-G131H-N243V, A088T-N109G-G131H-N243V-L257G,     A088T-N109G-G131H-N243V-S248N-K256R,     A088T-N109G-G131H-N243V-S248N-L257G, A088T-N109G-G131H-S248N-L257G,     A088T-N109G-G131H-T158S-L257G,     A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R,     A088T-N109G-G131H-T158S-N243V, A088T-N109G-G131H-T158S-N243V-K256R,     A088T-N109G-G131H-T158S-N243V-K256R-L257G,     A088T-N109G-G131H-T158S-N243V-L257G, A088T-N109G-N218S-K256R,     A088T-N109G-N218S-N243V-S248N-L257G,     A088T-N109G-N218S-S248N-K256R-L257G,     A088T-N109G-N243V-S248N-K256R-L257G,     A088T-N109G-N243V-S248N-L257G-I268V, A088T-N109G-S248N-K256R-L257G,     A088T-N109G-T158S-N218S-K256R, A088T-N109G-T158S-N218S-N243V-L257G,     A088T-N109G-T158S-N243V-K256R-I268V,     A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N218S-N243V,     A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-S248N,     A088T-N218S-S248N-L257G, A088T-N243V-K256R, A088T-N243V-L257G,     A088T-S145T-T158S-S248N, A088T-T158S-L257G,     A088T-T158S-N218S-S248N-L257G, A088T-T158S-N243V-K256R-L257G-Q271H,     A088T-T158S-S248N, A088T-V143A-T158S-K256R, A116T-G131H-K256R,     A116T-G131H-N218S, A116T-G131H-N243V, A116T-G131H-N243V-K256R,     A116T-G131H-N243V-L257G, A116T-G131H-S248N-K256R,     A116T-G131H-T158S-N218S-I234T-N243V-S248N-K256R,     A116T-G131H-T158S-N243V-L257G, A116T-G131H-T158S-N243V-S248N-K256R,     A116T-G131H-V143F-T158S-N218S, A116T-L257G, A116T-N218S,     A116T-N218S-L257G, A116T-N218S-N243V-L257G, A116T-N243V-K256R,     A116T-N243V-S248N, A116T-N243V-S248N-K256R-L257G, A116T-S248N,     A116T-T158S-N218S-N243V, A116T-T158S-N218S-S248N, A116T-T158S-N243V,     A116T-T158S-N243V-K256R, A116T-T158S-N243V-L257G,     A116T-T158S-N243V-S248N, A116T-T158S-S248N-K256R-L257G,     A116T-V149I-T158S-N243V-S248N-K256R-Q271H, G131H-N218S-N243V-L257G,     G131H-N243V, G131H-N243V-S248N-K256R, G131H-T158S,     G131H-T158S-N218S-N243V-K256R, G131H-T158S-N243V-K256R-L257G,     G131H-T158S-N243V-S248N-L257G, N109G-A116T-G131H-N218S-K256R-L257G,     N109G-A116T-G131H-N218S-L257G,     N109G-A116T-G131H-N218S-N243V-K256R-L257G,     N109G-A116T-G131H-N218S-S248N-K256R, N109G-A116T-G131H-N243V-K256R,     N109G-A116T-G131H-N243V-L257G,     N109G-A116T-G131H-N243V-S248N-K256R-L257G, N109G-A116T-G131H-S248N,     N109G-A116T-G131H-S248N-I268V,     N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R,     N109G-A116T-G131H-T158S-N218S-S248N-L257G,     N109G-A116T-G131H-T158S-S248N, N109G-A116T-G131H-T158S-S248N-K256R,     N109G-A116T-N218S, N109G-A116T-N218S-N243V-K256R,     N109G-A116T-N218S-N243V-K256R-L257G, N109G-A116T-N218S-S248N-L257G,     N109G-A116T-N243V-K256R, N109G-A116T-N243V-S248N-K256R-L257G,     N109G-A116T-S248N-L257G, N109G-A116T-T158S-G211V-N243V-S248N-K256R,     N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S,     N109G-A116T-T158S-N218S-N243V-K256R-L257G,     N109G-A116T-T158S-N218S-N243V-L257G,     N109G-A116T-T158S-N218S-N243V-S248N-L257G,     N109G-A116T-T158S-N218S-S248N-K256R-L257G, N109G-A116T-T158S-N243V,     N109G-A116T-T158S-Q275R, N109G-G131H-A137V-T158S-N218S-S248N,     N109G-G131H-N218S-K237N, N109G-G131H-N218S-N243V-K256R-L257G,     N109G-G131H-N218S-S248N-K256R, N109G-G131H-N243V-K256R-L257G,     N109G-G131H-S145F-N218S-N243V-K256R-L257G, N109G-G131H-S248N-K256R,     N109G-G131H-S248N-L257G, N109G-G131H-T158S-K256R,     N109G-G131H-T158S-N218S-N243V-K256R, N109G-G131H-T158S-N243V,     N109G-G131H-T158S-N243V-K256R-L257G, N109G-G131H-T158S-N243V-L257G,     N109G-G131H-T158S-S248N-L257G, N109G-G131H-T158S-S248N-Q271R,     N109G-N218S-L257G, N109G-N218S-N243V, N109G-N243V-K256R-L257G,     N109G-N243V-S248N-K256R-L257G, N109G-T158S-I268V, N109G-T158S-K256R,     N109G-T158S-N218S-N243V-K256R-L257G, N109G-T158S-N218S-S248N-L257G,     N109G-T158S-N243V, N109G-T158S-N243V-K256R-L257G,     N109G-T158S-N243V-S248N, N109S-A116T-S248N, N218S,     N218S-N243V-S248N-K256R-L257G, N218S-S248N-L257G, N243V-K256R,     N243V-S248N-K256R, N243V-S248N-K256R-L257G,     S105P-A116T-T158S-N218S-N243V-S248N-K256R, S248N, T158S-N243V-K256R,     T158S-N243V-L257G, S018F-S162L, S018P-D120N, P014T-S037T,     S009T-K141R, S161P-S162L, N61P-S63G-N109Q-A128S-S224A-N243V,     A88T-N109G-A114S-A116T-A128S-N243V,     A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A128S-S183V,     N109M-A128S-S224A, A88T-N109S-A116T-A128S-S224A-N243V,     N109Q-A128S-S224A-N243V, A88T-N109M-A116T-A128S-S224A-N243V,     S63G-A128S, N109S-A128S-S224A-N243V, A88T-N109G-A116T-N243V,     N61S-N109G-N243V, N101Q-N109Q-A128S-S224A-N243V,     A88T-N109G-A116T-T158S-N243V-K256R, N109G-A116T, A88T-N109G,     N61G-N109G-N243V, N109G-A128S-P129S-S130T-S224A-N243V,     A88T-N109Q-A116T-A128S-S224A-N243V, N062L-S063N-Q217L,     N062S-S063R-Q217E, N062S-S063L-Q217L, S063G, S063G-Q217L, S063N and     S063N-Q217L.

In one aspect of said composition, said variant of a parent protease, said parent protease having at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2, comprises the amino acid substitutions: S024G+S053G+S078N+S101N+G128A/S+Y217Q/L/D; and optionally one or more groups of subsitutions selected from the following groups of mutations: A088T+N109G+A116T+G131H+N243V+L257G, S033T+N076D, S009T+N109G+K141R+N243V, S162G+K256R, N109G+A116T, N109G+L257G, S162G+L257G, N061G+N109G+N243V, N109G+N243V+S248A, S033T+N076D+N109G+N218S+N243V+S248N+K256R, N109G+A116T+N243V+K256R, A088T+N109G+A116T+G131H+N243V, A088T+N109G, N109G+N243V, T158S+L257G, N061S+N109G+N243V, P040A+N109G+N243V+S248N+K256R, S009T+S018T+Y021N+N109G+K141R, A088T+N109G+A116T+T158S+N243V+K256R, A088T+N109G+A116T+T158S+N218S+L257G, N109G+K256R, N109G+N243V+K256R, S063G+K256R, S063G+N109G, S063G, S063G+N076D, S033T+N076D+N218S and N076D+N218S.

In one aspect of said composition, said protease is a variant of a parent protease having SEQ ID NO:6, said variant comprising one or more of the following groups of amino acid substitutions: A088T+N109G+A116T+G131H+N243V+L257G, S033T+N076D, S009T+N109G+A128S+K141R+N243V, S162G+K256R, N109G+A116T, N109G+L257G, S162G+L257G, N061G+N109G+N243V, N109G+A128S+N243V+S248A, S033T+N076D+N109G+A128S+N218S+N243V+S248N+K256R, N109G+A116T+N243V+K256R, A088T+N109G+A116T+G131H+N243V, A088T+N109G, N109G+N243V, T158S+L257G, N061S+N109G+N243V, P040A+N109G+A128S+N243V+S248N+K256R, S009T+S018T+Y021N+N109G+A128S+K141R, A088T+N109G+A116T+T158S+N243V+K256R, A088T+N109G+A116T+T158S+N218S+L257G, N109G+K256R, N109G+A128S+N243V+K256R, S063G+K256R, S063G+N109G, S063G+A128S, S063G+N076D, S033T+N076D+A128S+N218S and N076D+N218S.

In one aspect of said composition, said parent protease is a subtilisin protease.

In one aspect of said composition, said parent protease is a subtilisin protease selected from the group consisting of B. amyloliquefaciens subtilisin protease BPN′ (SEQ ID NO:2), Geobacillus stearothermophilus (formerly classified as B. stearothermophilus), B. subtilis, B. licheniformis, B. lentus, B. brevis, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.

In one aspect of said composition, said cold water protease is a variant of a parent protease, said cold water protease comprising a total of three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected from groups (a) and (b) below, wherein at least one mutation is selected from group (a):

-   a) 1, 9, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 33, 43, 76, 102,     109, 137, 141, 158, 169, 204, 210, 218, 243, 248, 249, 256, 257,     260, and 269; and -   b) 24, 25, 40, 52. 53, 55, 58, 59, 61, 62, 63, 68, 78, 86, 87, 88,     89, 92, 96, 97, 100, 101, 103, 104, 106, 111, 114, 115, 116, 117,     118, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133, 134, 144,     145, 159, 161, 162, 167, 194, 203, 206, 213, 217, 227, 232, 239,     240, 242, 265, 267, and 275.

In one aspect of said composition, said variant of a parent protease, said parent protease having SEQ ID NO:2, comprises a total of three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected from groups (a) and (b) below, wherein at least one mutation is selected from group (a):

-   (a) A1E/T, S9/T, P14L, A15L, L16C, H17T, S18L/T, Q19K, G20A, Y21N/T,     T22L, G23E, S33T, K43Y, N76D, G102A, N109A/S/G, A137V, K141R, T158S,     G169A, S204E, P210S, N218S, N243P/V, S248N/A, S249A, K256R, L257G,     S260P, and N269D; and -   (b) S24G/R/E, N25G, P40A/E, P52L, S53G, T55P, F58G, Q59S,     N61E/P/G/S, N62Q/R/S, S63G/H, V68A, S78N, P86S, S87D/G, A88T/V,     S89Y, A92G, L96T, G97A, G100N/Q/T, S101N, Q103E/H, Y104N, W106F,     I111V, A114G, I115V, A116N/T, N117S, N118G, N123A/G/Q/V, M124I/V,     S125A, L126A, G128A/S, P129E/Q/S/V, S130G, G131S/H, S132N, A133V,     A134T, A144K, S145D, S159K, S161P, S162G/K, Y167A, P194L, V203Y,     Q206D/E, K213L, Y217Q/L/D, V227T, A232T, P239R/V, N240K, T242R,     K265N, L267V, and Q275E.

In one aspect of said composition, said variant of a parent protease has a total net charge of −1, 0 or +1 relative to the BPN′ wild-type.

In one aspect of said composition, said composition comprises an adjunct ingredient selected from the group consisting of: a surfactant, a builder, a chelating agent, a dye transfer inhibiting agent, a dispersant, one or more additional enzymes, an enzyme stabilizer, a catalytic material, a bleach activator, a hydrogen peroxide, a source of hydrogen peroxide, a preformed peracid, a polymeric dispersing agent, a clay soil removal/anti-redeposition agent, a brightener, a suds suppressor, a dye, a perfume, a perfume delivery system, a structure elasticizing agent, a fabric softener, a carrier, a hydrotrope, a processing aid, a solvent, a pigment and mixtures thereof.

In one aspect of said composition, said composition comprises a material selected from the group consisting of an encapsulate comprising a perfume, a hueing agent, an amphiphilic cleaning polymer, a brightener, a chelating agent and mixtures thereof.

In one aspect of said composition, said composition comprises a second non-immunoequivalent protease selected from the group comprising:

-   a) subtilisins (EC 3.4.21.62); -   b) trypsin-like or chymotrypsin-like proteases; -   c) metalloproteases; and -   d) mixtures thereof.

In one aspect of said composition, said composition comprises a second non-immunoequivalent protease that is a subtilisin (EC 3.4.21.62) protease said subtilisin (EC 3.4.21.62) protease being a cold water GG36 variant protease.

In one aspect of said composition, said composition comprises an additional enzyme selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, cellobiose dehydrogenases, xyloglucanases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, or mixtures thereof.

In one aspect of said composition, said composition comprises an additional enzyme is selected from the group consisting of:

-   a) first-wash lipases; -   b) alpha-amylases that are greater than 90% identical to the     wild-type alkaline alpha-amylases derived from Bacillus sp. NCIB     12289, NCIB 12512, NCIB 12513, DSM 9375 DSM 12368, DSMZ no. 12649,     KSM AP1378, KSM K36 or KSM K38; -   c) microbial-derived endoglucanases; and -   d) mixtures thereof.

In one aspect of said composition, said composition comprises a material selected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant; -   b) a chelant selected from the group consisting of DTPA (Diethylene     triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),     DTPMP (Diethylene triamine penta(methylene phosphonic acid)),     ethylenediaminedisuccinic acid (EDDS),     1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and     derivatives of such chelants; and -   c) mixtures thereof.

In one aspect of said composition, said composition comprises a material selected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant having an alkyl chain length     of from 10 to 14 and a degree of ethoxylation from 1 to 4; -   b) a chelant selected from the group consisting of DTPA (Diethylene     triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),     DTPMP (Diethylene triamine penta(methylene phosphonic acid)),     ethylenediaminedisuccinic acid (EDDS),     1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and     derivatives of such chelants; and -   c) mixtures thereof.

In one aspect of said composition, said composition comprises, based on total composition weight, a chelant level of from about 1% to about 5%.

In one aspect of said composition, said composition comprises a fabric hueing agent selected from the group consisting of

-   a) dyes; -   b) dye-clay conjugates comprising at least one cationic-basic dye     and a smectite clay; and -   c) mixtures thereof.

In one aspect of said composition, said composition comprises a fabric hueing agent selected from the group consisting of

-   a) small molecule dyes; polymeric dyes and mixtures thereof; -   b) dye-clay conjugates comprising at least one cationic-basic dye     and a smectite clay; and -   c) mixtures thereof.

In one aspect of said composition, said composition comprises, based on total composition weight:

-   a) from about 0.0005 wt % to about 0.1 wt % of said cold water     protease; and -   b) one or more of the following -   (i) from about 0.00003 wt % to about 0.1 wt % fabric hueing agent; -   (ii) from about 0.001 wt % to about 5 wt %, perfume capsules and/or -   (iii) from about 0.1 wt % to about 5 wt % amphiphilic cleaning     polymer.

In one aspect of said composition, said composition has a single or multi-compartment unit dose form.

In one aspect of said composition, said composition is in the form of a multi-compartment unit dose, wherein at least one protease is in a different compartment to any additional enzymes and/or chelant.

In one aspect, a composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof said composition being a fabric and home care product is disclosed.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said cold water protease is a variant of the protease having SEQ ID NO:2 (subtilisin BPN′) said variant comprising one or more of the following sets of mutations, insertions or deletions: S182E, N109I, N109D-Y217L-S248R, N109D-S188R-Y217L, S87D-Y217L-S248R, S87R-N109D-Y217L-S248R, S87R-N109D-S188D-Y217L-S248R, G128A-Y217Q, I111V-M124V, M124V-Y217Q, N62Q-G97A, S89Y-M124V, V68A, V68A-A92G, V68A-G97A, V68A-I111V, V68A-S89Y, V68A-V227T, V68A-Y217Q, W106F-Y217Q, G97A-G128A-Y217Q, G97A-L126A-Y217Q, G97A-M124V-L126A-Y217Q, G97A-N123G-Y217Q, L96T-G97A-Y217Q, M124V-L126A-Y217Q, N62Q-G128A-Y217Q, N62Q-G97A-Y217Q, G97N-G128A-Y217M, G97G-G128S-Y217E, G97A-G128A-Y217Q, G97M-G128S-Y217E, G97A-G128S-Y217Q, G97D-G128S-Y217Q, G97M-G128G-Y217M, G97G-G128S-Y217Q, G97S-G128S-Y217Q, G97G-G128A-Y217Q, G97S-G128A-Y217E, G97A-G128S-Y217L, G97A-G128A-Y217N, G97Q-G128S-Y217L, G97A-G128A-Y217M, G97A-G128A-Y217S, G97D-G128A-Y217Q, G97M-G128S-Y217Q, G97Q-G128G-Y217D-S87Y, G97S-G128A-Y217N, G97A-G128S-Y217T, G97D-G128S-Y217E, G97D-G128A-Y217L, G97G-G128S-Y217E-S78P-A272T, G97T-G128S-Y217D, G97D-G128A-Y217I, G97Q-G128S-Y217Q, G97G-G128A-Y217D, G97Q-G128A-Y217N, G97S-G128A-Y217M, G97S-G128S-Y217N, G97S-G128S-Y217M, G97E-G128S-Y217M, G97S-G128P-Y217Q, G97T-G128S-Y217Q, G97D-G128S-Y217Q-A73T, G97E-G128S-Y217N, G97G-G128A-Y217I, G97Q-G128A-Y217D, G97Q-G128S-Y217M, G97R-G128T-Y217Q-S162P, G97S-G128S-Y217D, G97T-G128P-Y217I, G97Q-G128G-Y217E, G97C-G128G-Y217N, G97D-G128S-Y217H, G97M-G128S-Y217L, G97M-G128S-Y217N, G97S-G128S-Y217E, G97M-G128S-Y217I, G97A-G128P-Y217A, G97R-G128S-Y217D, G97A-G128A-Y217Q-S145D, G97A-G128A-Y217Q-P239R, G97A-G128A-Y217Q-N61E-P129E-S162K-K213L-N240K, G97A-G128A-Y217Q-N61E, G97A-G128A-Y217Q-P40E-A144K-K213L, G97A-G128A-Y217Q-P129E, G97A-G128A-Y217Q-N61E-P129E-S159K, G97A-G128A-Y217Q-K213L, G97A-G128A-Y217Q-S87D, G97A-G128A-Y217Q-Q206E, G97A-G128A-Y217Q-S24R-P40E-S145D-S159K-K213L, G97A-G128A-Y217Q-K265N, G97A-G128A-Y217Q-S24R, G97A-G128A-Y217Q-P40E, G97A-G128A-Y217Q-Q275E, G97A-G128A-Y217Q-P129E-S145D-N240K, G97A-G128A-Y217Q-A144K, G97A-G128A-Y217Q-S159K, G97A-G128A-Y217Q-S162K, G97A-G128A-Y217Q-N240K, G97A-G128A-Y217Q-S53G, G97A-G128A-Y217Q-S78N, G97A-G128A-Y217Q-S53G-S78N, G97A-G128A-Y217Q-S53G-I111V, G97A-G128A-Y217Q-S53G-N117S, G97A-G128A-Y217Q-S53G-S132N, G97A-G128A-Y217Q-Y104N-S132N, G97A-G128A-Y217Q-S53G-S78N-I111V, G97A-G128A-Y217Q-S53G-S78N-N117S, G97A-G128A-Y217Q-S53G-S78N-S132N, G97A-G128A-Y217Q-S53G-Y104N-S132N, G97A-G128A-Y217Q-S78N-Y104N-S132N, Y217L-V068C-A069G, Y217L-I079F-A098G, Y217L-P086T-S101D-Q103S-V147I, Y217L-A088T-P129S-G146D, Y217L-V093I-G128D-P129R, Y217L-Z096.01D-A098R, Y217L-Z096.01H-A098G, Y217L-G097S-Z097.01S-A098G-A273T, Y217L-A098S-D099G-G100D, Y217L-Z098.01N, Y217L-D099G-Z099.01N, Y217L-D099G-Z099.01S, Y217L-D099V-S101D, Y217L-Z099.01S, Y217L-G100D, Y217L-S101D-Q103H, Y217L-S101G-A151V, Y217L-S101H-G102S, Y217L-S101H-Q103D, Y217L-G102R-Q103C-Y104C-V192I, Y217L-Q103D, Y217L-V121I-I122S-N123C, Y217L-V121L-N123C, Y217L-I122S-N123S, Y217L-M124I, Y217L-M124V, Y217L-L126F-P129Z-S182N, Y217L-L126Y, Y217L-G127S-P129D, Y217L-Z127.01N-G128S-P129S, Y217L-G128H-P129Y, Y217L-G128S-P129D, Y217L-G128S-P129D-S248R, Y217L-G128S-P129G, Y217L-P129G-G131Z, Y217L-P129G-S130H-S132Z, Y217L-P129H-G131Z, Y217L-P129L, Y217L-P129S-S130H-S132Z, Y217L-P129Z, Y217L-P129Z-S130G, Y217L-P129Z-S130G-G131H-S132H, Y217L-P129Z-S130H, Y217L-S130V-G131D-S132I, S87T-A88L-S89G-G97A-G128A-Y217Q, N61P-S63H-G97A-G128A-Y217Q, S87G-A88V-S89A-G97A-G128A-Y217Q, P86S-S87G-A88V-G97A-G128A-Y217Q, Q59S-N61P-G97A-G128A-Y217Q, S24G-N25G-G97A-G128A-Y217Q, N61P-N62S-G97A-G128A-Y217Q, G97A-G128A-P129Q-S130G-G131S-Y217Q, L75S-N76Y-G97A-G128A-Y217Q, G97A-G128A-V203Y-Y217Q, T55P-G97A-G128A-Y217Q, A88V-L90I-G97A-G128A-Y217Q, G97A-G128A-G211R-N212S-K213V-Y217Q, G23A-S24G-N25G-G97A-G128A-Y217Q, T22N-S24A-G97A-G128A-Y217Q, S24R-G97A-G128A-Y217Q, G97A-A98S-G128A-Y217Q, G97A-G128A-T158G-S159G-Y217Q, Q59E-N61P-G97A-G128A-Y217Q, G97A-A98E-G128A-Y217Q, G97A-G128A-Y217Q-P86S-S87G-A88V-A116N-N117S-N118G, G97A-G128A-Y217Q-S63T-P86S-S87G-A88V, G97A-G128A-Y217Q-P86S-S87G-A88V-P239R, G97A-G128A-Y217Q-S24G-N25G-N61P-N62S-P194L-A232T, G97A-G128A-Y217Q-P129Q-S130G-G131S-A133V-L267V, G97A-G128A-Y217Q-A134T-L267V, G97A-G128A-Y217Q-S24R-P40E-P129E-S159K-K265R, G97A-G128A-Y217Q-A134T-G211T, G97A-G128A-Y217Q-S24R-P129E, G97A-G128A-Y217Q-I111V-S161P, G97A-G128A-Y217Q-T55P-P129Q, G97A-G128A-Y217Q-I115V-L267V, G97A-G128A-Y217Q-P86S-S87G-A88V-A116S-N117G-N118R, G97A-G128A-Y217Q-V203Y-L267V, G97A-G128A-Y217Q-S24G-N25G-S78N-S101N-V203Y, G97A-G128A-Y217Q-P52S-T55P-V203Y, G97A-G128A-Y217Q-Q59S-N61P-A116S-N117G-N118R, G97A-G128A-Y217Q-S24G-N25G-P129Q-S130G-G131S, G97A-G128A-Y217Q-P86S-S87G-A88V-T242R, G97A-G128A-Y217Q-P40E-T55P-N269K, G97A-G128A-Y217Q-G23A-S24G-N25G-A116N-N117S-N118G, G97A-G128A-Y217Q-V8L-N25Y-P129Q-S130G-G131S, G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-G211T-L267V, G97A-G128A-Y217Q-S24R-A116N-N117S-N118G, G97A-G128A-Y217Q-S24R-A128S-P129G, G97A-G128A-Y217Q-P129Q-S130G-G131S-N240K, G97A-G128A-Y217Q-N25Y-P129Q-S130G-G131S, G97A-G128A-Y217Q-S87T-A88L-S89G-A134T, G97A-G128A-Y217Q-P129Q-S130G-G131S-L267V, G97A-G128A-Y217Q-S87G-A88V-S89A-A116N-N117S-N118G, G97A-G128A-Y217Q-N61P-P129Q-S130G-G131S, G97A-G128A-Y217Q-N61P-S78N-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-T55P-P129V-P194S, G97A-G128A-Y217Q-T55P-P129V, G97A-G128A-Y217Q-S24G-N25G-T55P-S78N-S101N, G97A-G128A-Y217Q-T55P-S78N-I115V, G97A-G128A-Y217Q-N25Y-S87G-A88V-S89A, G97A-G128A-Y217Q-A134T-N240K, G97A-G128A-Y217Q-S24R-Q59S-N61P, G97A-G128A-Y217Q-G23A-S24G-N25G-P239R, G97A-G128A-Y217Q-T55P-A116S-N117G-N118R, G97A-G128A-Y217Q-A134T-S161P, G97A-G128A-Y217Q-S24G-N25G-S53G-N61P-S101N-V203Y, G97A-G128A-Y217Q-N25Y-Q59S-N61P, G97A-G128A-Y217Q-N25Y-P129Q-S130G-G131S-A137T, G97A-G128A-Y217Q-G23A-S24G-N25G-N61P-S63H, G97A-G128A-Y217Q-T55P-N61P-S78N-S101N-V203Y, G97A-G128A-Y217Q-P129Q-N240K, G97A-G128A-Y217Q-T55P-A134T, G97A-G128A-Y217Q-N25Y-N61P-S63H, G97A-G128A-Y217Q-S87T-A88L-S89G-P129S, G97A-G128A-Y217Q-T55P-L75H-N76G, G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-T55P-I115V, G97A-G128A-Y217Q-T55P-A116N-N117S-N118G, G97A-G128A-Y217Q-S24G-N25G-A116N-N117S-N118G, G97A-G128A-Y217Q-S24R-P129Q-S130G-G131S, G97A-G128A-Y217Q-G23A-S24G-N25G-G211R-N212S-K213V, G97A-G128A-Y217Q-S24G-N25G-T55P-N61P-S78N-S101N-V203Y, G97A-G128A-Y217Q-T55P-S78N-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-I115V-A273S, G97A-G128A-Y217Q-N25Y-T55P, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-Q59S-N61P-N240K, G97A-G128A-Y217Q-S161P-L267V, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S78N-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-S24G-N25G-N61P-S101N, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S101N-V203Y, G97A-G128A-Y217Q-N240K, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-S24G-N25G-T55P-S101N, G97A-G128A-Y217Q-N61P-S63H-A128S-P129Q, G97A-G128A-Y217Q-S89Y-P129Q-S130G-G131S, G97A-G128A-Y217Q-P129Q-S130G-G131S-V203Y, G97A-G128A-Y217Q-I115V-N240K, G97A-G128A-Y217Q-S53G-N61P-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-S161P-V203Y, G97A-G128A-Y217Q-S87T-A88L-S89G-N240K, G97A-G128A-Y217Q-S87T-A88L-S89G-P239R, G97A-G128A-Y217Q-T55P-N61P-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-S24G-N25G-I115V-A134T, G97A-G128A-Y217Q-Y6Q-P129Q-S130G-G131S, G97A-G128A-Y217Q-T55P-S78N-S89Y, G97A-G128A-Y217Q-S24G-N25G-T55P-S78N-A88V-S101N, G97A-G128A-Y217Q-N61P-S63H-S78N-I111V-A134T, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S78N-S101N, G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S101N-V203Y, G97A-G128A-Y217Q-S53G-N61P-S101N-V203Y, G97A-G128A-Y217Q-S53G-T55P-S78N-S101N-V203Y, G97A-G128A-Y217Q-S53G-T55P-N61P-S78N-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-N240K-A273S, G97A-G128A-Y217Q-S78N-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-Q59S-N61P-S87T-A88L-S89G, G97A-G128A-Y217Q-N61P-S63H-S78N-S161P, G97A-G128A-Y217Q-N61P-S63H-S78N-I111V, G97A-G128A-Y217Q-T55P-A128S-P129Q, G97A-G128A-Y217Q-N61P-S78N-S101N-V203Y, G97A-G128A-Y217Q-N61E-A144K, G97A-G128A-Y217Q-A134T-P239R, G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S101N-V203Y, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S101N-V203Y, G97A-G128A-Y217Q-N61P-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-T55P-N240K, G97A-G128A-Y217Q-S24G-N25G-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-P129Q-S130G-G131S-P239R, G97A-G128S-Y217Q, G97A-G128A-Y217Q-S53G-N61P-S101N, G97A-G128A-Y217Q-I111V-P129Q-G211T, G97A-G128A-Y217Q-S24G-N25G-S53G-S101N-V203Y, G97A-G128A-Y217Q-Q59S-N61P-S87G-A88V-S89A, G97A-G128A-Y217Q-S24G-N25G-S78N-S87T-A88L-S89G-S101N, G97A-G128A-Y217Q-P129Q-S130G-G131S-S162K, G97A-G128A-Y217Q-T55P-P129Q-S130G-G131S, G97A-G128A-Y217Q-T55P-V203Y, G97A-G128A-Y217Q-S87G-A88V-S89A-P129Q-S130G-G131S, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-N61P-S78N-S87T-A88L-S89G, G97A-G128A-Y217Q-I111V-P129Q-S130G-G131S, G97A-G128A-Y217Q-I111V-A273S, G97A-G128A-Y217Q-N61P-S87T-A88L-S89G, G97A-G128A-Y217Q-T22N-S24A-N61P-S63H, G97A-G128A-Y217Q, G97A-G128A-Y217Q-S53G-S78N-S87T-A88L-S89G-S101N-P129S-V203Y, G97A-G128A-Y217Q-S159K-L267V, G97A-G128A-Y217Q-P40E-S53Y-S78Y-P86S-S87G-A88V, G97A-G128A-Y217Q-S24R-S145D, G97A-G128A-Y217Q-I111V-S159K, G97A-G128A-Y217Q-T55P-P129L, G97A-G128A-Y217Q-Q59S-N61P-V203Y, G97A-G128A-Y217Q-T55P-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S101N, G97A-G128A-Y217Q-S53G-N61P-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-S89Y, G97A-G128A-Y217Q-S24R-P129V, G97A-G128A-Y217Q-S87G-A88V-S89A-A116N-N117S-N118G-P172H, G97A-G128A-Y217Q-I111V-A134T, G97A-G128A-Y217Q-Q59S-N61P-P129Q-S130G-G131S, G97A-G128A-Y217Q-P5S-S87G-A88V-S89A-A116G-N117R, Y217Q-N61P-A97G-G102A-A128G-P129S, G97A-G128A-Y217Q-S24G-N25G-S53G-N61P-S78N, G97A-G128A-Y217Q-S145D-S159K-N240K-Q275E, G97A-G128A-Y217Q-T55P-A128S-P129D, G97A-G128A-Y217Q-G23A-S24G-N25G-A128S-P129D, G97A-G128A-Y217Q-S24G-N25G-S53G-S78N-S87T-A88L-S89G-V203Y, G97A-G128A-Y217Q-I111V-P239R, G97A-G128A-Y217Q-S87G-A88V-S89A-S162K, G97A-G128A-Y217Q-S87T-A88L-S89G-I115V, G97A-G128A-Y217Q-S24G-N25G-T55P-S78N, G97A-G128A-Y217Q-T55P-A92G, G97A-G128A-Y217Q-S24G-N25G-S53G-S87T-A88L-S89G-V203Y, G97A-G128A-Y217Q-T22N-S24A-T55P, G97A-G128A-Y217Q-S53G-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-S24G-N25G-S53G-T55P-S78N-S87T-A88L-S89G, G97A-G128A-Y217Q-P129Q-S130G-G131S-S159K, G97A-Y217Q-N61P-N62Q-G100N-A128G, G97A-G128A-Y217Q-S24R-S78N-S182P-L267V, G97A-G128A-Y217Q-P239R-A273S, G97A-G128A-Y217Q-S53G-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-P129Q-S130G-G131S-T242R, G97A-G128A-Y217Q-S3F-S87T-A88L-S89G-G211T, G97A-G128A-Y217Q-S24G-N25G-L75H-N76G, G97A-G128A-Y217Q-S53G-T55P-N61P-S78N-S87T-A88L-S89G, G97A-G128A-Y217Q-S87T-A88L-S89G-A144K, G97A-G128A-Y217Q-S78N-S87T-A88L-S89G-V203Y, G97A-G128A-Y217Q-Q59S-N61P-A116N-N117S-N118G, G97A-G128A-Y217Q-S87T-A88L-S89G-I111V, G97A-G128A-Y217Q-S24R-S145D-P239R-Q275E, G97A-G128A-Y217Q-S145D-A273S, G97A-G128A-Y217Q-S24G-N25G-K141E-T242R, G97A-G128A-Y217Q-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-A116N-N117S-N118G-P129Q-S130G-G131S, G97A-G128A-Y217Q-S89Y-G211T, G97A-G128A-Y217Q-S87G-A88V-S89A-A116N-N117S-N118G-A144T, G97A-G128A-Y217Q-S24G-N25G-S78N-S87T-A88L-S89G-S101N-V203Y, G97A-G128A-Y217Q-S24G-N25G-P129V, G97A-Y217Q-N61P-A128G-P129S-S130P, G97A-G128A-Y217Q-T55P-N61P-S87T-A88L-S89G-G110C-S130P, G97A-Y217Q-N123G-A128G, G97A-G128A-Y217Q-N61P-N62Q-G100N-G102A-M124I, S78N-G97A-G128A-Y217Q, G97A-S101N-G128A-Y217Q, G97A-G128A-A137V-Y217Q, N61P-G97A-G128A-Y217Q, G97A-G128A-S130P-Y217Q, G97A-Q103N-G128A-Y217Q, S63T-G97A-G128A-Y217Q, G97A-G102A-G128A-Y217Q, G97A-N109D-G128A-Y217Q-S248R, S87R-G97A-G128A-Y217Q, G97A-G128A-S188D-Y217Q, S87D-G97A-G128A-Y217Q-S248R, G97A-G128A-S188D-S248R-Y217Q, G97A-G128A-S248D-Y217Q, S78N-G97A-G128A-Y217Q-L267V, S78N-G97A-G128A-Y217Q-S161P, S78N-G97A-G128A-Y217Q-1115V, S78N-G97A-G128A-Y217Q-A273S, S78N-G97A-G128A-Y217Q-G211T, S78N-G97A-G128A-Y217Q, S78N-G97A-G128A-Y217Q-I111V, S78N-G97A-G128A-Y217Q-V147L, S78N-G97A-G128A-Y217Q-I108V, S78N-G97A-G128A-Y217Q-S89Y, S78N-G97A-G128A-Y217Q-A138T, G97A-G128A-Y217Q-A134T-K213L, G97A-G128A-Y217Q-G23A-S24G-N25G-P129V, G97A-G128A-Y217Q-S24R-P239R, G97A-G128A-Y217Q-S24R-S87T-A88L-S89G

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said variant of the protease having SEQ ID NO:2 comprises three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations within the group of positions comprising positions 24, 25, 40, 52. 53, 55, 58, 59, 61, 62, 63, 68, 78, 86, 87, 88, 89, 92, 96, 97, 100, 101, 103, 104, 106, 111, 114, 115, 116, 117, 118, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133, 134, 144, 145, 159, 161, 162, 167, 194, 203, 206, 213, 217, 227, 232, 239, 240, 242, 265, 267, and 275.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said variant of the protease having SEQ ID NO:2 comprises a total of three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected from groups (a) and (b) below:

-   (a) charged mutations selected from the group consisting of N61E,     A144K, P129E, P239R, P40E, Q103E, Q206E, Q275E, S145D, S159K, S162K,     S24R, S63H, S87D and T242R; and -   (b) neutral mutations selected from the group consisting of A114G,     A116N, A133V, A134T, A232T, A88V, A92G, F58G, G100T, G128A, G131S,     G97A, I111V, I115V, K213L, K265N, L126A, L267V, L96T, M124V, N117S,     N118G, N123G, N240K, N25G, N61P, N62Q, N62R, N62S, P129Q, P129V,     P194L, P239V, P52L, P86S, Q59S, S101N, S125A, S130G, S132N, S161P,     S24G, S53G, S78N, S87G, S89Y, T55P, V203Y, V227T, V68A, W106F,     Y104N, Y167A,and Y217Q.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said variant of the protease having SEQ ID NO:2 comprises one or more mutations, and having a total net charge of −1, 0 or +1 relative to the BPN′ wild-type.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises an adjunct ingredient selected from the group consisting of: a surfactant, a builder, a chelating agent, a dye transfer inhibiting agent, a dispersant, one or more additional enzyme, an enzyme stabilizer, a catalytic material, a bleach activators, a hydrogen peroxide, a source of hydrogen peroxide, a preformed peracid, a polymeric dispersing agent, a clay soil removal/anti-redeposition agent, a brightener, a suds suppressor, a dye, a perfume, a perfume delivery system, a structure elasticizing agent, a fabric softener, a carrier, a hydrotrope, a processing aid, a solvent, a pigment and mixtures thereof.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a third non-immunoequivalent protease that is a subtilisin (EC 3.4.21.62) protease said subtilisin (EC 3.4.21.62) protease being a cold water GG36 variant protease.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises an additional enzyme selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, cellobiose dehydrogenases, xyloglucanases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, or mixtures thereof.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises an additional enzyme is selected from the group consisting of:

-   a) first-wash lipases; -   b) alpha-amylases that are greater than 90% identical to the     wild-type alkaline alpha-amylases derived from Bacillus sp. NCIB     12289, NCIB 12512, NCIB 12513, DSM 9375 DSM 12368, DSMZ no. 12649,     KSM AP1378, KSM K36 or KSM K38; -   c) microbial-derived endoglucanases; and -   d) mixtures thereof.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a material selected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant; -   b) a chelant selected from the group consisting of DTPA (Diethylene     triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),     DTPMP (Diethylene triamine penta(methylene phosphonic acid)),     ethylenediaminedisuccinic acid (EDDS),     1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and     derivatives of such chelants; and -   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a material selected from the group consisting of:

-   a) an alcohol ethoxysulphate surfactant having an alkyl chain length     of from 10 to 14 and a degree of ethoxylation from 1 to 4; -   b) a chelant selected from the group consisting of DTPA (Diethylene     triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),     DTPMP (Diethylene triamine penta(methylene phosphonic acid)),     ethylenediaminedisuccinic acid (EDDS),     1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and     derivatives of such chelants; and -   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises, based on total composition weight, a chelant level of from about 1% to about 5%.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a fabric hueing agent selected from the group consisting of

-   a) dyes; -   b) dye-clay conjugates comprising at least one cationic-basic dye     and a smectite clay; and -   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a fabric hueing agent selected from the group consisting of

-   a) small molecule dyes; polymeric dyes and mixtures thereof; -   b) dye-clay conjugates comprising at least one cationic-basic dye     and a smectite clay; and -   c) mixtures thereof.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises based on total composition weight:

-   a) from about 0.0005 wt % to about 0.1 wt % of said cold water     protease; and -   b) one or more of the following:     -   (i) from about 0.00003 wt % to about 0.1 wt % of a fabric hueing         agent;     -   (ii) from about 0.001 wt % to about 5 wt %, of a perfume         capsules;     -   (iii) from about 0.1 wt % to about 5 wt % of a amphiphilic         cleaning polymer;     -   (iv) from about 0.00003 wt % to about 0.1 wt % of a GG36 variant         protease.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition has a single or multi-compartment unit dose form.

In one aspect of said composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition is in the form of a multi-compartment unit dose, wherein at least one protease is in a different compartment to any additional enzymes and/or chelant.

A fabric and home product that may comprise one or more cold water proteases and a material selected from the group consisting of: an encapsulate comprising a perfume, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, with the balance of any aspects of the aforementioned composition is made up of one or more adjunct materials, is disclosed.

In one aspect of the aforementioned fabric and home care product, said fabric and home care product may comprise, based on total fabric and home care product weight, from about 0.0005 wt % to about 0.1 wt %, from about 0.001 wt % to about 0.05 wt %, or even from about 0.002wt % to about 0.03wt % of said cold water protease.

In one aspect of the aforementioned fabric and home care product, said fabric and home care product may comprise, based on total fabric and home care product weight, about 0.00003wt % to about 0.1wt %, from about 0.00008wt % to about 0.05wt %, or even from about 0.0001wt % to about 0.04wt %, fabric hueing agent;

In one aspect of the aforementioned fabric and home care product, said fabric and home care product may comprise, based on total fabric and home care product weight, from about 0.001wt % to about 5wt %, from about 0.01wt % to about 2wt %, or even from about 0.03wt % to about 0.5wt %, perfume capsules.

In one aspect of the aforementioned fabric and home care product, said fabric and home care product may comprise, based on total fabric and home care product weight, from about 0.1 wt % to about 5 wt %, from about 0.25 wt % to about 2.5 wt %, or even from about 0.3 wt % to about 1.5 wt % amphiphilic cleaning polymer.

Cold Water Proteases

In addition to the cold water proteases disclosed above in the Fabric and Home Care Products, suitable cold water protease variants are enzymes that exhibit one or more of the following four criteria:

-   -   (a) a performance index of at least 1.1, at least 1.2, at least         1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at         least 1.8, at least 1.9; at least 2 from 1.1 to about 10, from         1.1 to about 8 or even from 1.1 to about 5 on BMI at pH 8 and         60° F. when compared to Purafect Prime (SEQ ID NO:2 with the         mutation Y217L), as defined in Test Method 2 and/or Test Method         3;     -   (b) a performance index of at least 1.3, at least 1.4, at least         1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at         least 2, from 1.3 to about 10, from 1.3 to about 8 or even from         1.3 to about 5 on BMI at pH 8 and 60° F. when compared to BPN′         (SEQ ID NO:2), as defined in Test Method 2 and/or Test Method 3;     -   (c) a performance index of at least 0.9, at least 1.0, at least         1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at         least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2         from 0.9 to about 10, from 0.9 to about 8 or even from 0.9 to         about 5 on BMI at pH 8 and 60° F. when compared to SEQ ID NO:4,         as defined in Test Method 2 and/or Test Method 3;     -   (d) a performance index of at least 0.9, at least 1.0, at least         1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at         least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2         from 0.9 to about 10, from 0.9 to about 8 or even from 0.9 to         about 5 on BMI at pH 8 and 60° F. when compared to SEQ ID NO:6,         as defined in Test Method 2 and/or Test Method 3.

Examples of such proteases can be found in Examples 2-18 and 31 of the present specification. Cold water proteases as defined above may be used in Fabric and Home Care Products. Thus, in one aspect, Fabric and Home Care Products comprising such cold water proteases are disclosed.

GG36 Proteases of this Invention

Suitable GG36 proteases can be selected from the variants described in Examples 19 to 30. In one aspect, GG36 cold water proteases may be Series 1 GG36 cold water proteases.

Suitable Series 1 GG36 cold water protease variants include enzymes derived from a parent protease, said parent protease's sequence being at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence of SEQ ID NO:755, said variant having one or more of the following characteristics:

-   -   a) a Test Method 4 performance index of at least 1.1, at least         1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at         least 1.7, at least 1.8, at least 1.9; at least 2; from 1.1 to         about 10, from 1.1 to about 8 or even from 1.1 to about 5;     -   b) a Test Method 5 performance index of at least 1.1, at least         1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at         least 1.7, at least 1.8, at least 1.9, at least 2; from 1.1 to         about 10, from 1.1 to about 8 or even from 1.1 to about 5;     -   c) a Test Method 6 performance index of at least 1.0, at least         1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at         least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2,         from 1.0 to about 10, from 1.0 to about 8 or even from 1.0 to         about 5.     -   Suitable Series 1 GG36 cold water proteases can be derived from         subtilisins, particularly those derived from subtilisin Bacillus         Lentus of SEQ ID NO:755 and in one aspect may comprise one or         more of the following mutations:     -   A1R, Q2S, V4R, V4S, S9A, R10S, P14K, A16S, H17R, N18R, G20R,         T22A, T22R, S24R, S24W, G25R, G25V, V26F, L42I, N43R, N43A,         G46R, P52F, P52E, P52N, T57R, Q59A, N62E, N62Q, V68A, V68C,         T71G, I72C, A74C. L75A, L75F, L75R, N76D, S78R, L82R, P86W,         E89P, E89T, E89G, E89H, E89I, E89V, E89W, Y91N, K94N, G100S,         S101A, S101N, S101G, S101D, S103G, S103N, V104L, V104I, S106V,         S106G, A108I, L111V, E112V, G115K, G115R, N117F, G118I, V121F,         S128D, S128F, S128L, S128N, P129E, S144R, L148I, A158E. G159E,         S160D, S166D, N185E, N185I, R186H, S188E, S188D, D197F, V203E,         Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G, P210R, S212I,         S212F, Y214F, A215N, A215D, A215E, L217E, L217N, T224A, A230E,         A231I, Q236F, N238R, N238K, P239K, P239G, P239R, P239S, W241R,         S242R, S242L, N243R, V244R, N248I, N248V, H249R, L250I, N252R,         T253R, L262D, Y263F, S265F, L267V, L267N. N269I, N269R, E271F,         E271I, E271H, E271P, E271T, E271V, E271L and/or A272F     -   In one aspect, suitable Series 1 GG36 cold water proteases         include subtilisins, particularly Bacillus Lentus of SEQ ID         NO:755, that may comprise one or more of the following sets of         mutations, insertions or deletions:     -   T022R-S024R, S009A-E271L, N018R-W241R, N018R-G115R, N043R-H249R,         G020R-H249R, V004R-H249R, G020R-S024R, N018R-H249R, S009A-G020R,         G020R-W241R, S009A-S078R, G020R-G115R, N018R-S024R, S024R-S242R,         T022R-G115R, N018R-N043R, G020R-N043R, N018R-S242R, S242R-N269R,         N018R-V244R, S024R-N269R, G020R-E271L, S024R-E271L, V004R-S009A,         G020R-N269R, A001R-S024R, V244R-E271L, S009A-N018R, W241R-E271L,         V004R-S024R, S009A-H249R, S009A-T022R, N062E-P129E, N062E-G159E,         A016S-L148I, A158E-H249R, A016S-N062E, L111V-S188D, T022A-N062E,         N062E-L148I, T022A-P129E, N062E-E271F, N062E-A158E, A016S-G159E,         N062E-R186H, S128N-G159E, N062E-S188D, N062E-S128N, L148I-G159E,         S103G-A158E, L111V-G159E, A158E-E271F, A016S-S188D, T022A-L111V,         S128N-A158E, A016S-A158E, V104L-A158E, S128N-R186H, G159E-Y209E,         N062E-S101A, L111V-Y209E, L148I-S188D, S101A-Y209E, T022A-S188D,         A016S-T022A, S128N-P129E, A016S-Y209E, A016S-S128N, T022A-E089P,         S128N-Y209E, E089P-A158E, N062E-S103G, R186H-E271F, A016S-P129E,         E089P-G159E, L111V-H249R, S101A-P129E, L148I-Y209E, T022A-G159E,         P129E-H249R, P129E-Y209E, V104L-P129E, S128N-S188D, L111V-A158E,         T022A-A158E, N062E-Y209E, N062E-H249R, S101A-R186H, E089P-P129E,         P129E-E27I, T22A-L111V-G159E, S101A-S103G-V104L-Y209E,         S101A-S103G-V104L-G159E, S101A-S103G-V104L-S188D,         S101G-S103A-V104I-G159D, T22A-S103G-G159E,         T22A-S128N-E271F-Y209E, T22A-Y209E-E271F, T22A-S101A-Y209E,         S101A-Y209E-E271F, T22A-L111V-S128N, T22A-S101A-G159E,         S101A-S103G-V104L, T22A-S101A-S103G-V104L, S101A-S103G-V104L,         S101G-S103A-V104I, S101A-S103G-V104L-S128N,         S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,         S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,         S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K,         S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K,         S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K,         S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K,         S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K,         S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K,         S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D,         N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,         N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,         T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-T253R,         S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R,         T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-G159E-A232V-Q245R-N248D-H249R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N238R,         S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N76D, and/or         S101G-S103A-V104I-G159E-A232V-Q245R-N248D-E271F.     -   In one aspect, suitable Series 1 GG36 cold water proteases         include variants of subtilisins, particularly Bacillus Lentus of         SEQ ID NO:755, said variants comprising three, four, five, six,         seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations within         the group of positions comprising positions 1, 2, 4, 9, 10, 14,         16, 17, 18, 20, 22, 24, 25, 26, 42, 43, 46, 52, 57, 59, 62, 68,         71, 72, 74, 75, 76, 78, 82, 86, 89, 91, 94, 100, 101, 103, 104,         106, 108, 111, 112, 115, 117, 118, 121, 128, 129, 144, 148, 158,         159, 160, 166, 185, 186, 188, 197, 203, 209, 210, 212, 214, 215,         217, 224, 230, 231, 236, 238, 239, 241, 242, 243, 244, 248, 249,         250, 252, 253, 262, 263, 265, 267, 269, 271 and 272.     -   In one aspect, suitable Series 1 GG36 cold water proteases         include variants of subtilisins, particularly Bacillus Lentus of         SEQ ID NO:755, said variants comprising a total of three, four,         five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15         mutations selected from the group comprising: A1R, Q2S, V4R,         V4S, S9A, R10S, P14K, A16S, H17R, N18R, G20R, T22A, T22R, S24R,         S24W, G25R, G25V, V26F, L42I, N43R, N43A, G46R, P52F, P52E,         P52N, T57R, Q59A, N62E, N62Q, V68A, V68C, T71G, I72C, A74C.         L75A, L75F, L75R, N76D, S78R, L82R, P86W, E89P, E89T, E89G,         E89H, E89I, E89V, E89W, Y91N, K94N, G100S, S101A, S101N, S101G,         S101D, S103G, S103N, V104L, V104I, S106V, S106G, A108I, L111V,         E112V, G115K, G115R, N117F, G118I, V121F, S128D, S128F, S128L,         S128N, P129E, S144R, L148I, A158E. G159E, S160D, S166D, N185E,         N185I, R186H, S188E, S188D, D197F, V203E, Y209S, Y209N, Y209F,         Y209T, Y209E, Y209H, Y209G, P210R, S212I, S212F, Y214F, A215N,         A215D, A215E, L217E, L217N, T224A, A230E, A231I, Q236F, N238R,         N238K, P239K, P239G, P239R, P239S, W241R, S242R, S242L, N243R,         V244R, N248I, N248V, H249R, L250I, N252R, T253R, L262D, Y263F,         S265F, L267V, L267N, N269I, N269R, E271F, E271I, E271H, E271P,         E271T, E271V, E271L and A272F; and optionally one or more of the         following mutations: S103A, G159D, Q236H, Q245R, N248D and         N252K.

In one aspect, said Series 1 GG36 cold water protease is variant of subtilisin GG36 having SEQ ID NO:755, said variant comprising one or more mutations, and having a total net charge of −1, −4, −3, −2, −1 or 0 relative to subtilisin GG36 wild-type, is disclosed.

In one aspect, said Series 1 GG36 cold water proteases are low ionic strength cold water proteases. Such low ionic strength proteases are variants of subtilisin GG36 having SEQ ID NO:755, said variants comprising one or more mutations, and having a total net charge of −5, −4, −3, −2, −1 or 0 relative to subtilisin GG36 wild-type, is disclosed. These mutations are selected from:

-   -   (a) two or more of the following mutations: A1R, Q2S, V4R, V4S,         S9A, R10S, P14K, A16S, T22A, T22R, S24R, G25V, V26F, L42I, P52F,         P52E, P52N, N62E, N62Q, V68A, V68C, T71G, I72C, A74C, L75A,         L75F, S78R, E89P, E89T, E89G, E89H, E89W, Y91N, K94N, G100S,         S101A, S101N, S101G, S101D, S103G, S103N, V104L, V104I, A108I,         L111V, E112V, G115K, N117F, V121F, S128D, S128F, S128L, S128N,         P129E, L148I, A158E. G159E, S160D, S166D, N185E, R186H, S188E,         S188D, V203E, Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G,         P210R, S212I, S212F, Y214F, A215N, A215D, A215E, L217E, L217N,         T224A, A230E, A231I, Q236F, N238R, N238K, P239K, P239G, P239R,         N248V, H249R, L250I, L262D, Y263F, S265F, L267V, L267N. N269I,         N269R, E271F, E271I, E271H and A272F; and/or     -   (b) one or more of the following sets of mutations: N062E-P129E,         N062E-G159E, A016S-L148I, A158E-H249R, A016S-N062E, L111V-S188D,         T022A-N062E, N062E-L148I, T022A-P129E, N062E-E271F, N062E-A158E,         A016S-G159E, N062E-R186H, S128N-G159E, N062E-S188D, N062E-S128N,         L148I-G159E, S103G-A158E, L111V-G159E, A158E-E271F, A016S-S188D,         T022A-L111V, S128N-A158E, A016S-A158E, V104L-A158E, S128N-R186H,         G159E-Y209E, N062E-S101A, L111V-Y209E, L148I-S188D, S101A-Y209E,         T022A-S188D, A016S-T022A, S128N-P129E, A016S-Y209E, A016S-S128N,         T022A-E089P, S128N-Y209E, E089P-A158E, N062E-S103G, R186H-E271F,         A016S-P129E, E089P-G159E, L111V-H249R, S101A-P129E, L148I-Y209E,         T022A-G159E, P129E-H249R, P129E-Y209E, V104L-P129E, S128N-S188D,         L111V-A158E, T022A-A158E, N062E-Y209E, N062E-H249R, S101A-R186H,         E089P-P129E, P129E-E271F, T22A-L111V-G159E,         S101A-S103G-V104L-Y209E, S101A-S103G-V104L-G159E,         S101A-S103G-V104L-S188D, S101G-S103A-V104I-G159D,         T22A-S103G-G159E, T22A-S128N-E271F-Y209E, T22A-Y209E-E271F,         T22A-S101A-Y209E, S101A-Y209E-E271F, T22A-L111V-S128N,         T22A-S101A-G159E, S101A-S103G-V104L, T22A-S101A-S103G-V104L,         S101A-S103G-V104L, S101G-S103A-V104I, S101A-S103G-V104L-S128N,         S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,         S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K,         S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K,         S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K,         S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K,         S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K,         S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K,         S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K,         S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D,         N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,         N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,         T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-T253R,         S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R,         T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-G159E-A232V-Q245R-N248D-H249R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N238R,         S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N76D and         S101G-S103A-V104I-G159E-A232V-Q245R-N248D-E271F;

In one aspect the above low ionic strength Series 1 GG36 cold water proteases form part of a detergent composition that is diluted in water, typically within a washing machine, to form a wash liquor, whose conductivity is from about 0.1 mS/cm to about 3 mS/cm, from about 0.3 mS/cm to about 2.5 mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm

In one aspect, said Series 1 GG36 cold water proteases are high ionic strength cold water proteases. Such high ionic strength proteases are variants of subtilisin GG36 having SEQ ID NO:755, said variants comprising two or more mutations, and having a total net charge of +5, +4, +3, +2, +1 or 0 relative to subtilisin GG36 wild-type. These mutations are selected from:

-   -   a) two or more of the following mutations V4R, H17R, N18R, G20R,         T22R, S24R, S24W, G25R, N43R, N43A, G46R, P52F, P52N, T57R,         Q59A, N62Q, T71G, L75R, N76D, S78R, L82R, P86W, E89P, E89W,         E89T, E89I, E89H, E89V, V104L, S106V, S106G, G115R, G118I,         V121F, S144R, N185I, D197F, Y209N, Y209S, L217E, A231I, P239R,         P239S, W241R, S242R, S242L, N243R, V244R, N248I, H249R, N252R,         T253R, E271T, E271V, E271L, E271H, E271F, E271P, A1R, S9A, S212F         and N269R; and/or     -   b) one or more of the following sets of mutations T022R-S024R,         S009A-E271L, N018R-W241R, N018R-G115R, N043R-H249R, G020R-H249R,         V004R-H249R, G020R-S024R, N018R-H249R, S009A-G020R, G020R-W241R,         S009A-S078R, G020R-G115R, N018R-S024R, S024R-S242R, T022R-G115R,         N018R-N043R, G020R-N043R, N018R-S242R, S242R-N269R, N018R-V244R,         S024R-N269R, G020R-E271L, S024R-E271L, V004R-S009A, G020R-N269R,         A001R-S024R, V244R-E271L, S009A-N018R, W241R-E271L, V004R-S024R,         S009A-H249R, S009A-T022R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F,         S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R,         S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R,         S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K,         S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K

In one aspect, the above high ionic strength Series 1 GG36 cold water proteases form part of a detergent composition that is diluted in water, typically within a washing machine, to form a wash liquor, whose conductivity is from about 3 mS/cm to about 30 mS/cm, from about 3.5 mS/cm to about 20 mS/cm, or even from about 4 mS/cm to about 10 mS/cm.

The charge of the Series 1 GG36 cold water protease variants is expressed relative to subtilisin GG36 protease wild-type having the amino acid sequence of SEQ ID NO:755. The amino acids that impart a single negative charge are D and E and those that impart a single positive charge are R, H and K. Any amino acid change versus SEQ ID NO:755 that changes a charge is used to calculate the charge of the Series 1 GG36 cold water protease variant. For example, introducing a negative charge mutation from a wild-type neutral position will add a net charge of −1 to the Series 1 GG36 cold water protease variant, whereas introducing a negative charge mutation (D or E) from a wild-type positive amino acid residue (R, H or K) will add a net charge of −2. Summing the charge changes from all the amino acid residues that are different for the Series 1 GG36 cold water protease variant versus subtilisin GG36 protease wild-type having the amino acid sequence of SEQ ID NO:755 gives the charge change of the Series 1 GG36 cold water protease variant. Without wishing to be bound by theory, it is believed that:

(a) the preferred charge range for Series 1 GG36 cold water proteases to be used in low conductivity laundry detergent solutions is −5, −4, −3, −2, −1, 0, particularly −2, −1;

(b) the preferred charge range for Series 1 GG36 cold water proteases to be used in high conductivity laundry detergent solutions is +5, +4, +3, +2, +1, 0, particularly +2, +1. By correctly selecting the charge unexpectedly improved levels of cold water cleaning performance can be obtained.

Low conductivity solutions are defined as having a conductivity of from about 0.1 mS/cm to about 3 mS/cm, from about 0.3 mS/cm to about 2.5 mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm.

High conductivity having conductivity solutions are defined as having a conductivity of from about 3 mS/cm to about 30 mS/cm, from about 3.5 mS/cm to about 20 mS/cm, or even from about 4 mS/cm to about 10 mS/cm

The above examples should be viewed as non-limiting. Once mutations are combined to optimize cold water performance, the enzyme charge can also be balanced by mutations in further positions.

Protease Amino Acid Numbering, Enzyme Nomenclature and Additional Definitions

The numbering of amino acid positions used in this patent is the Bacillus amyloliquefaciens subtilisin BPN′ numbering system. Each amino acid position of each protease variant, including each Series 1 GG36 cold water protease variant, is numbered according to the numbering of corresponding amino acid position in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ as determined by alignment of the variant protease amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN′ amino acid sequence.

An alternative numbering scheme is numbering the specific amino acid sequence of the B. lentus subtilisin GG36 protease, having the amino acid sequence of SEQ ID NO:755. None of the amino acid positions of the protease variants, including the Series 1 GG36 cold water protease variants, described herein are numbered using this alternative numbering scheme.

In describing Series 1 GG36 cold water protease variants herein, the following nomenclature is used for ease of reference: Original amino acid(s):position(s):substituted amino acid(s).

Mutations are named by the one letter code for the parent amino acid, followed by a three digit position number and then the one letter code for the variant amino acid. For example, mutating glycine (G) at position 87 to serine (S) is represented as “G087S” or “G875”. Multiple mutations are indicated by inserting a “-” between the mutations. Mutations at positions 87 and 90 are represented as either “G087S-A090Y” or “G87S-A90Y” or “G87S+A90Y” or “G087S+A090Y”. For deletions, the one letter code “Z” is used. For an insertion relative to the parent sequence, the one letter code “Z” is on the left side of the position number. For a deletion, the one letter code “Z” is on the right side of the position number. For insertions, the position number is the position number before the inserted amino acid(s), plus 0.01 for each amino acid. For example, an insertion of three amino acids alanine (A), serine (S) and tyrosine (Y) between position 87 and 88 is shown as “Z087.01A-Z087.02S-Z087.03Y.” Thus, combining all the mutations above plus a deletion at position 100 is: “G087S-Z087.01A-Z087.02S-Z087.03Y-A090Y-A100Z.”

In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed. The single letter X refers to any of the twenty amino acids.

The term “wild-type” in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is native or naturally occurring sequence. As used herein, the term “naturally-occurring” refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that are found in nature (i.e., have not been manipulated by means of recombinant methods). As used herein, the term, “non-naturally occurring” refers to anything that is not found in nature (e.g., recombinant nucleic acids produced in the laboratory).

As used herein with regard to amino acid residue positions, “corresponding to” or “corresponds to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide. As used herein, “corresponding region” generally refers to an analogous position along related proteins or a reference protein.

The terms “derived from” and “obtained from” refer to not only a protease produced or producible by a strain of the organism in question, but also a protease encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protease which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protease in question. To exemplify, “proteases derived from Bacillus” refers to those enzymes having proteolytic activity which are naturally-produced by Bacillus, as well as to serine proteases like those produced by Bacillus sources but which through the use of genetic engineering techniques are produced by non-Bacillus organisms transformed with a nucleic acid encoding the serine proteases.

The term “identical” in the context of two nucleic acids or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence, as measured using one of the following sequence comparison or analysis algorithms.

As used herein, “homologous genes” refers to a pair of genes from different, but usually related species, which correspond to each other and which are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthologous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes).

The term “mature” form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide without the signal peptide sequence and propeptide sequence.

As used herein, “homology” refers to sequence similarity or identity, with identity being preferred. Homology may be determined using standard techniques known in the art (See e.g., Smith and Waterman, Adv. Appl. Math. 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol. 48:443 [1970\; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; software programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis.); and Devereux et al., Nucl. Acid Res. 12:387-395 [1984]). One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (See, Feng and Doolittle, J. Mol. Evol. 35:351-360 [1987]). The method is similar to that described by Higgins and Sharp (See, Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al., (See, Altschul et al., J. Mol. Biol. 215:403-410 [1990]; and Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). A particularly useful BLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth. Enzymol. 266:460-480 [1996]). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity.

The percent sequence identity between a reference sequence and a test sequence of interest may be readily determined by one skilled in the art. The percent identity shared by polynucleotide or polypeptide sequences is determined by direct comparison of the sequence information between the molecules by aligning the sequences and determining the identity by methods known in the art. An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, (See, Altschul, et al., J. Mol. Biol., 215:403-410 [1990]). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1992]) alignments (B) of 50, expectation (E) of 10, M′5, N′−4, and a comparison of both strands.

The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (See e.g., Karlin and Altschul, supra). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a serine protease nucleic acid of this invention if the smallest sum probability in a comparison of the test nucleic acid to a serine protease nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Where the test nucleic acid encodes a serine protease polypeptide, it is considered similar to a specified serine protease nucleic acid if the comparison results in a smallest sum probability of less than about 0.5, and more preferably less than about 0.2.

Percent “identical” or “identity” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same or have a specified percentage of nucleic acid residues or amino acid residues, respectively, that are the same, when compared and aligned for maximum similarity, as determined using a sequence comparison algorithm or by visual inspection. “Percent sequence identity” or “% identity” or “% sequence identity or “% amino acid sequence identity” of a subject amino acid sequence to a reference (i.e., query) amino acid sequence means that the subject amino acid sequence is identical (i.e., on an amino acid-by-amino acid basis) by a specified percentage to the query amino acid sequence over a comparison length when the sequences are optimally aligned. Thus, 80% amino acid sequence identity or 80% identity with respect to two amino acid sequences means that 80% of the amino acid residues in two optimally aligned amino acid sequences are identical.

“Percent sequence identity” or “% identity” or “% sequence identity or “% nucleotide sequence identity” of a subject nucleic acid sequence to a reference (i.e. query) nucleic acid sequence means that the subject nucleic acid sequence is identical (i.e., on a nucleotide-by-nucleotide basis for a polynucleotide sequence) by a specified percentage to the query sequence over a comparison length when the sequences are optimally aligned. Thus, 80% nucleotide sequence identity or 80% identity with respect to two nucleic acid sequences means that 80% of the nucleotide residues in two optimally aligned nucleic acid sequences are identical.

In some embodiments, the percent sequence identity or % sequence identity”or “% identity” of a subject sequence to a query sequence can be calculated by optimally aligning the two sequences and comparing the two optimally aligned sequences over the comparison length. The number of positions in the optimal alignment at which identical residues occur in both sequences are determined, thereby providing the number of matched positions, and the number of matched positions is then divided by the total number of positions of the comparison length (which, unless otherwise specified, is the length of the query sequence). The resulting number is multiplied by 100 to yield the percent sequence identity of the subject sequence to the query sequence.

“Optimal alignment” or “optimally aligned” refers to the alignment of two (or more) sequences giving the highest percent identity score. For example, optimal alignment of two protein sequences can be achieved by manually aligning the sequences such that the maximum number of identical amino acid residues in each sequence are aligned together or by using software programs or procedures described herein or known in the art. Optimal alignment of two nucleic acid sequences can be achieved by manually aligning the sequences such that the maximum number of identical nucleotide residues in each sequence are aligned together or by using software programs or procedures described herein or known in the art.

In some embodiments, two polypeptide sequences are deemed “optimally aligned” when they are aligned using defined parameters, such as a defined amino acid substitution matrix, gap existence penalty (also termed gap open penalty), and gap extension penalty, so as to achieve the highest similarity score possible for that pair of sequences. The BLOSUM62 scoring matrix (See, Henikoff and Henikoff, supra) is often used as a default scoring substitution matrix in polypeptide sequence alignment algorithms (e.g., BLASTP). The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each residue position in the gap. Exemplary alignment parameters employed are: BLOSUM62 scoring matrix, gap existence penalty=11, and gap extension penalty=1. The alignment score is defined by the amino acid positions of each sequence at which the alignment begins and ends (e.g., the alignment window), and optionally by the insertion of a gap or multiple gaps into one or both sequences, so as to achieve the highest possible similarity score.

Optimal alignment between two or more sequences can be determined manually by visual inspection or by using a computer, such as, but not limited to for example, the BLASTP program for amino acid sequences and the BLASTN program for nucleic acid sequences (See e.g., Altschul et al., Nucleic Acids Res. 25(17):3389-3402 (1997); See also, the National Center for Biotechnology Information (NCBI) website).

Suitable Fabric Hueing Agents

Fluorescent optical brighteners emit at least some visible light. In contrast, fabric hueing agents can alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes, dye-clay conjugates, and pigments that satisfy the requirements of Test Method 1 in the Test Method Section of the present specification. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example:

(1) Tris-Azo Direct Blue Dyes of the Formula

where at least two of the A, B and C napthyl rings are substituted by a sulfonate group, the C ring may be substituted at the 5 position by an NH₂ or NHPh group, X is a benzyl or naphthyl ring substituted with up to 2 sulfonate groups and may be substituted at the 2 position with an OH group and may also be substituted with an NH2 or NHPh group.

(2) Bis-Azo Direct Violet Dyes of the Formula:

where Z is H or phenyl, the A ring is preferably substituted by a methyl and methoxy group at the positions indicated by arrows, the A ring may also be a naphthyl ring, the Y group is a benzyl or naphthyl ring, which is substituted by sulfate group and may be mono or disubstituted by methyl groups.

(3) Blue or Red Acid Dyes of the Formula

where at least one of X and Y must be an aromatic group. In one aspect, both the aromatic groups may be a substituted benzyl or naphthyl group, which may be substituted with non water-solubilising groups such as alkyl or alkyloxy or aryloxy groups, X and Y may not be substituted with water solubilising groups such as sulfonates or carboxylates. In another aspect, X is a nitro substituted benzyl group and Y is a benzyl group

(4) Red Acid Dyes of the Structure

where B is a naphthyl or benzyl group that may be substituted with non water solubilising groups such as alkyl or alkyloxy or aryloxy groups, B may not be substituted with water solubilising groups such as sulfonates or carboxylates.

(5) Dis-Azo Dyes of the Structure

wherein X and Y, independently of one another, are each hydrogen, C₁-C₄ alkyl or C₁-C₄-alkoxy, Rα is hydrogen or aryl, Z is C₁-C₄ alkyl; C₁-C₄-alkoxy; halogen; hydroxyl or carboxyl, n is 1 or 2 and m is 0, 1 or 2, as well as corresponding salts thereof and mixtures thereof

(6) Triphenylmethane Dyes of the Following Structures

and mixtures thereof.

In another aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of Colour Index (Society of Dyers and Colourists, Bradford, UK) numbers 1,4-Naphthalenedione, 1-[2-[2-[4-[[4-(acetyloxy)butyl]ethylamino]-2-methylphenyl]diazenyl]-5-nitro-3-thienyl]-Ethanone, 1-hydroxy-2-(1-naphthalenylazo)-Naphthalenedisulfonic acid, ion(2-), 1-hydroxy-2-[[4-(phenylazo)phenyl]azo]-Naphthalenedisulfonic acid, ion(2-), 2-[(1E)-[4-[bis(3-methoxy-3-oxopropyl)amino]-2-methylphenyl]azo]-5-nitro-3-Thiophenecarboxylic acid, ethyl ester, 2-[[4-[(2-cyanoethyl)ethylamino]phenyl]azo]-5-(phenylazo)-3-Thiophenecarbonitrile, 2-[2-[4-[(2-cyanoethyl)ethylamino]phenyl]diazenyl]-5-[2-(4-nitrophenyl)diazenyl]-3-Thiophenecarbonitrile, 2-hydroxy-1-(1-naphthalenylazo)-Naphthalenedisulfonic acid, ion(2-), 2-hydroxy-1-[[4-(phenylazo)phenyl]azo]-Naphthalenedisulfonic acid, ion(2-), 4,4′-[[4-(dimethylamino)-2,5-cyclohexadien-1-ylidene]methylene]bis[N,N-dimethyl-Benzenamine, 6-hydroxy-5-[(4-methoxyphenyl)azo]-2-Naphthalenesulfonic acid, monosodium salt, 6-hydroxy-5-[(4-methylphenyl)azo]-2-Naphthalenesulfonic acid, monosodium salt, 7-hydroxy-8-[[4-(phenylazo)phenyl]azo]-1,3-Naphthalenedisulfonic acid, ion(2-), 7-hydroxy-8-[2-(1-naphthalenyl)diazenyl]-1,3-Naphthalenedisulfonic acid, ion(2-), 8-hydroxy-7-[2-(1-naphthalenyl)diazenyl]-1,3-Naphthalenedisulfonic acid, ion(2-), 8-hydroxy-7-[2-[4-(2-phenyldiazenyl)phenyl]diazenyl]-1,3-Naphthalenedisulfonic acid, ion(2-), Acid Black 1, Acid black 24, Acid Blue 113, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid blue 29, Acid blue 3, Acid blue 40, Acid blue 45, Acid blue 62, Acid blue 7, Acid blue 75, Acid Blue 80, Acid Blue 83, Acid blue 9, Acid Blue 90, Acid green 27, Acid orange 12, Acid orange 7, Acid red 14, Acid red 150,Acid red 151, Acid red 17, Acid red 18, Acid red 266, Acid red 27, Acid red 4, Acid red 51, Acid red 52, Acid red 73, Acid red 87, Acid red 88, Acid red 92, Acid red 94, Acid red 97, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid violet 43, Acid Violet 49, Basic blue 159, Basic blue 16, Basic blue 22, Basic blue 3, Basic blue 47, Basic blue 66, Basic blue 75, Basic blue 9, Basic violet 1, Basic violet 2, Basic violet 3, Basic violet 4, Basic violet 10, Basic violet 35, C.I. Acid black 1, C.I. Acid Blue 10, C.I. Acid Blue 113, C.I. Acid Blue 25, C.I. Acid Blue 29, C.I. Acid Blue 290 C.I. Acid Red 103, C.I. Acid red 150, C.I. Acid red 52, C.I. Acid red 73, C.I. Acid red 88, C.I. Acid red 91, C.I. Acid violet 17, C.I. Acid violet 43, C.I. Direct Blue 1, C.I. Direct Blue 120, C.I. Direct Blue 34, C.I. Direct Blue 70, C.I. Direct Blue 71, C.I. Direct Blue 72, C.I. Direct Blue 82, C.I. Direct violet 51, C.I. Disperse Blue 10, C.I. Disperse Blue 100, C.I. Disperse Blue 101, C.I. Disperse Blue 102, C.I. Disperse Blue 106:1, C.I. Disperse Blue 11, C.I. Disperse Blue 12, C.I. Disperse Blue 121, C.I. Disperse Blue 122, C.I. Disperse Blue 124, C.I. Disperse Blue 125, C.I. Disperse Blue 128, C.I. Disperse Blue 130, C.I. Disperse Blue 133, C.I. Disperse Blue 137, C.I. Disperse Blue 138, C.I. Disperse Blue 139, C.I. Disperse Blue 142, C.I. Disperse Blue 146, C.I. Disperse Blue 148, C.I. Disperse Blue 149, C.I. Disperse Blue 165, C.I. Disperse Blue 165:1, C.I. Disperse Blue 165:2, C.I. Disperse Blue 165:3, C.I. Disperse Blue 171, C.I. Disperse Blue 173, C.I. Disperse Blue 174, C.I. Disperse Blue 175, C.I. Disperse Blue 177, C.I. Disperse Blue 183, C.I. Disperse Blue 187, C.I. Disperse Blue 189, C.I. Disperse Blue 193, C.I. Disperse Blue 194, C.I. Disperse Blue 200, C.I. Disperse Blue 201, C.I. Disperse Blue 202, C.I. Disperse Blue 205, C.I. Disperse Blue 206, C.I. Disperse Blue 207, C.I. Disperse Blue 209, C.I. Disperse Blue 21, C.I. Disperse Blue 210, C.I. Disperse Blue 211, C.I. Disperse Blue 212, C.I. Disperse Blue 219, C.I. Disperse Blue 220, C.I. Disperse Blue 222, C.I. Disperse Blue 224, C.I. Disperse Blue 225, C.I. Disperse Blue 248, C.I. Disperse Blue 252, C.I. Disperse Blue 253, C.I. Disperse Blue 254, C.I. Disperse Blue 255, C.I. Disperse Blue 256, C.I. Disperse Blue 257, C.I. Disperse Blue 258, C.I. Disperse Blue 259, C.I. Disperse Blue 260, C.I. Disperse Blue 264, C.I. Disperse Blue 265, C.I. Disperse Blue 266, C.I. Disperse Blue 267, C.I. Disperse Blue 268, C.I. Disperse Blue 269, C.I. Disperse Blue 270, C.I. Disperse Blue 278, C.I. Disperse Blue 279, C.I. Disperse Blue 281, C.I. Disperse Blue 283, C.I. Disperse Blue 284, C.I. Disperse Blue 285, C.I. Disperse Blue 286, C.I. Disperse Blue 287, C.I. Disperse Blue 290, C.I. Disperse Blue 291, C.I. Disperse Blue 294, C.I. Disperse Blue 295, C.I. Disperse Blue 30, C.I. Disperse Blue 301, C.I. Disperse Blue 303, C.I. Disperse Blue 304, C.I. Disperse Blue 305, C.I. Disperse Blue 313, C.I. Disperse Blue 315, C.I. Disperse Blue 316, C.I. Disperse Blue 317, C.I. Disperse Blue 321, C.I. Disperse Blue 322, C.I. Disperse Blue 324, C.I. Disperse Blue 328, C.I. Disperse Blue 33, C.I. Disperse Blue 330, C.I. Disperse Blue 333, C.I. Disperse Blue 335, C.I. Disperse Blue 336, C.I. Disperse Blue 337, C.I. Disperse Blue 338, C.I. Disperse Blue 339, C.I. Disperse Blue 340, C.I. Disperse Blue 341, C.I. Disperse Blue 342, C.I. Disperse Blue 343, C.I. Disperse Blue 344, C.I. Disperse Blue 345, C.I. Disperse Blue 346, C.I. Disperse Blue 351, C.I. Disperse Blue 352, C.I. Disperse Blue 353, C.I. Disperse Blue 355, C.I. Disperse Blue 356, C.I. Disperse Blue 357C.I. Disperse Blue 358, C.I. Disperse Blue 36, C.I. Disperse Blue 360, C.I. Disperse Blue 366, C.I. Disperse Blue 368, C.I. Disperse Blue 369, C.I. Disperse Blue 371, C.I. Disperse Blue 373, C.I. Disperse Blue 374, C.I. Disperse Blue 375, C.I. Disperse Blue 376, C.I. Disperse Blue 378, C.I. Disperse Blue 38, C.I. Disperse Blue 42, C.I. Disperse Blue 43, C.I. Disperse Blue 44, C.I. Disperse Blue 47, C.I. Disperse Blue 79, C.I. Disperse Blue 79:1, C.I. Disperse Blue 79:2, C.I. Disperse Blue 79:3, C.I. Disperse Blue 82, C.I. Disperse Blue 85, C.I. Disperse Blue 88, C.I. Disperse Blue 90, C.I. Disperse Blue 94, C.I. Disperse Blue 96, C.I. Disperse Violet 10, C.I. Disperse Violet 100, C.I. Disperse Violet 102, C.I. Disperse Violet 103, C.I. Disperse Violet 104, C.I. Disperse Violet 106, C.I. Disperse Violet 107, C.I. Disperse Violet 12, C.I. Disperse Violet 13, C.I. Disperse Violet 16, C.I. Disperse Violet 2, C.I. Disperse Violet 24, C.I. Disperse Violet 25, C.I. Disperse Violet 3, C.I. Disperse Violet 33, C.I. Disperse Violet 39, C.I. Disperse Violet 42, C.I. Disperse Violet 43, C.I. Disperse Violet 45, C.I. Disperse Violet 48, C.I. Disperse Violet 49, C.I. Disperse Violet 5, C.I. Disperse Violet 50, C.I. Disperse Violet 53, C.I. Disperse Violet 54, C.I. Disperse Violet 55, C.I. Disperse Violet 58, C.I. Disperse Violet 6, C.I. Disperse Violet 60, C.I. Disperse Violet 63, C.I. Disperse Violet 66, C.I. Disperse Violet 69, C.I. Disperse Violet 7, C.I. Disperse Violet 75, C.I. Disperse Violet 76, C.I. Disperse Violet 77, C.I. Disperse Violet 82, C.I. Disperse Violet 86, C.I. Disperse Violet 88, C.I. Disperse Violet 9, C.I. Disperse Violet 91, C.I. Disperse Violet 92, C.I. Disperse Violet 93, C.I. Disperse Violet 93:1, C.I. Disperse Violet 94, C.I. Disperse Violet 95, C.I. Disperse Violet 96, C.I. Disperse Violet 97, C.I. Disperse Violet 98, C.I. Disperse Violet 99, C.I. Reactive Black 5, C.I. Reactive Blue 19, C.I. Reactive Blue 4, C.I. Reactive Red 2, C.I. Solvent Blue 43, C.I. Solvent Blue 43, C.I. Solvent Red 14, C.I. Acid black 24, C.I. Acid blue 113, C.I. Acid Blue 29, C.I. Direct violet 7, C.I. Food Red 14, Dianix Violet CC, Direct blue 1, Direct Blue 71, Direct blue 75, Direct blue 78, Direct blue 80, Direct blue 279, Direct violet 11, Direct violet 31, Direct violet 35, Direct violet 48, Direct violet 5, Direct Violet 51, Direct violet 66, Direct violet 9, Disperse Blue 106, Disperse blue 148, Disperse blue 165, Disperse Blue 3, Disperse Blue 354, Disperse Blue 364, Disperse blue 367, Disperse Blue 56, Disperse Blue 77, Disperse Blue 79, Disperse blue 79:1, Disperse Red 1, Disperse Red 15, Disperse Violet 26, Disperse Violet 27, Disperse Violet 28, Disperse violet 63, Disperse violet 77, Eosin Y, Ethanol, 2,2′-[[4-[(3,5-dinitro-2-thienyl)azo]phenyl]imino]bis-, diacetate (ester), Lumogen F Blue 650, Lumogen F Violet 570, N-[2-[2-(3-acetyl-5-nitro-2-thienyl)diazenyl]-5-(diethylamino)phenyl]-Acetamide, N-[2-[2-(4-chloro-3-cyano-5-formyl-2-thienyl)diazenyl]-5-(diethylamino)phenyl]-Acetamide, N-[5-[bis(2-methoxyethyl)amino]-2-[2-(5-nitro-2,1-benzisothiazol-3-yl)diazenyl]phenyl]-Acetamide, N-[5-[bis[2-(acetyloxy)ethyl]amino]-2-[(2-bromo-4,6-dinitrophenyl)azo]phenyl]-Acetamide, Naphthalimide, derivatives, Oil Black 860, Phloxine B, Pyrazole, Rose Bengal, Sodium 6-hydroxy-5-(4-isopropylphenylazo)-2-naphthalenesulfonate, Solvent Black 3, Solvent Blue 14, Solvent Blue 35, Solvent Blue 58, Solvent Blue 59, Solvent Red 24, Solvent Violet 13, Solvent Violet 8, Sudan Red 380, Triphenylmethane, and Triphenylmethane, derivatives or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the group consisting of polymers containing conjugated chromogens (dye-polymer conjugates) and polymers with chromogens co-polymerized into the backbone of the polymer and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of fabric-substantive colorants sold under the name of Liquitint® (Milliken, Spartanburg, S.C., USA), dye-polymer conjugates formed from at least one reactive dye and a polymer selected from the group consisting of polymers comprising a moiety selected from the group consisting of a hydroxyl moiety, a primary amine moiety, a secondary amine moiety, a thiol moiety and mixtures thereof. In still another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of Liquitint® (Milliken, Spartanburg, S.C., USA) Violet CT, carboxymethyl cellulose (CMC) conjugated with a reactive blue, reactive violet or reactive red dye such as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme, Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product code S-ACMC, alkoxylated triphenyl-methane polymeric colourants, alkoxylated thiophene polymeric colourants, and mixtures thereof.

Suitable dye clay conjugates include dye clay conjugates selected from the group comprising at least one cationic/basic dye and a smectite clay, and mixtures thereof. In another aspect, suitable dye clay conjugates include dye clay conjugates selected from the group consisting of one cationic/basic dye selected from the group consisting of C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I. Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue 1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through 23, CI Basic Black 1 through 11, and a clay selected from the group consisting of Montmorillonite clay, Hectorite clay, Saponite clay and mixtures thereof. In still another aspect, suitable dye clay conjugates include dye clay conjugates selected from the group consisting of: Montmorillonite Basic Blue B7 C.I. 42595 conjugate, Montmorillonite Basic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I. 42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate, Montmorillonite Basic Red R1 C.I. 45160 conjugate, Montmorillonite C.I. Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate, Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3 C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate, Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black 2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite Basic Blue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555 conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite Basic Red R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate and mixtures thereof.

Suitable pigments include pigments selected from the group consisting of flavanthrone, indanthrone, chlorinated indanthrone containing from 1 to 4 chlorine atoms, pyranthrone, dichloropyranthrone, monobromodichloropyranthrone, dibromodichloropyranthrone, tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide, wherein the imide groups may be unsubstituted or substituted by C1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyl and heterocyclic radicals may additionally carry substituents which do not confer solubility in water, anthrapyrimidinecarboxylic acid amides, violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyanine which may contain up to 2 chlorine atoms per molecule, polychloro-copper phthalocyanine or polybromochloro-copper phthalocyanine containing up to 14 bromine atoms per molecule and mixtures thereof. In another aspect, suitable pigments include pigments selected from the group consisting of Ultramarine Blue (C.I. Pigment Blue 29), Ultramarine Violet (C.I. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (any mixture of fabric hueing agents can be used). Suitable fabric hueing agents can be purchased from Aldrich, Milwaukee, Wis., USA; Ciba Specialty Chemicals, Basel, Switzerland; BASF, Ludwigshafen, Germany; Dayglo Color Corporation, Mumbai, India; Organic Dyestuffs Corp., East Providence, R.I., USA; Dystar, Frankfurt, Germany; Lanxess, Leverkusen, Germany; Megazyme, Wicklow, Ireland; Clariant, Muttenz, Switzerland; Avecia, Manchester, UK and/or made in accordance with the examples contained herein.

Suitable hueing agents are described in more detail in U.S. Pat. No. 7,208,459 B2.

Encapsulates

In one aspect, an encapsulate comprising a core, a shell having an inner and outer surface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a material selected from the group consisting of perfumes; brighteners; dyes; insect repellants; silicones; waxes; flavors; vitamins; fabric softening agents; skin care agents in one aspect, paraffins; enzymes; anti-bacterial agents; bleaches; sensates; and mixtures thereof; and said shell may comprise a material selected from the group consisting of polyethylenes; polyamides; polystyrenes; polyisoprenes; polycarbonates; polyesters; polyacrylates; aminoplasts, in one aspect said aminoplast may comprise a polyureas, polyurethane, and/or polyureaurethane, in one aspect said polyurea may comprise polyoxymethyleneurea and/or melamine formaldehyde; polyolefins; polysaccharides, in one aspect said polysaccharide may comprise alginate and/or chitosan; gelatin; shellac; epoxy resins; vinyl polymers; water insoluble inorganics; silicone; and mixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamine formaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material and a shell, said shell at least partially surrounding said core material, is disclosed. At least 75%, 85% or even 90% of said encapsulates may have a fracture strength of from about 0.2 MPa to about 10 MPa, from about 0.4 MPa to about 5MPa, from about 0.6 MPa to about 3.5 MPa, or even from about 0.7 MPa to about 3MPa; and a benefit agent leakage of from 0% to about 30%, from 0% to about 20%, or even from 0% to about 5%.

In one aspect, at least 75%, 85% or even 90% of said encapsulates may have a particle size of from about 1 microns to about 80 microns, about 5 microns to 60 microns, from about 10 microns to about 50 microns, or even from about 15 microns to about 40 microns.

In one aspect, at least 75%, 85% or even 90% of said encapsulates may have a particle wall thickness of from about 30 nm to about 250 nm, from about 80 nm to about 180 nm, or even from about 100 nm to about 160 nm.

In one aspect, said encapsulates' core material may comprise a material selected from the group consisting of a perfume raw material and/or optionally a material selected from the group consisting of vegetable oil, including neat and/or blended vegetable oils including caster oil, coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, lemon oil and mixtures thereof; esters of vegetable oils, esters, including dibutyl adipate, dibutyl phthalate, butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate, trioctyl phosphate and mixtures thereof; straight or branched chain hydrocarbons, including those straight or branched chain hydrocarbons having a boiling point of greater than about 80° C.; partially hydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, including monoisopropylbiphenyl, alkylated naphthalene, including dipropylnaphthalene, petroleum spirits, including kerosene, mineral oil and mixtures thereof; aromatic solvents, including benzene, toluene and mixtures thereof; silicone oils; and mixtures there of.

In one aspect, said encapsulates' wall material may comprise a suitable resin including the reaction product of an aldehyde and an amine, suitable aldehydes include, formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycoluril, and mixtures thereof. Suitable melamines include, methylol melamine, methylated methylol melamine, imino melamine and mixtures thereof. Suitable ureas include, dimethylol urea, methylated dimethylol urea, urea-resorcinol, and mixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with the encapsulates, for example, in a capsule slurry and/or added to a fabric and home care product before, during or after the encapsulates are added to such fabric and home care product.

Suitable capsules that can be made by following the teaching of USPA 2008/0305982 A1; and/or USPA 2009/0247449 A1. Alternatively, suitable capsules can be purchased from Appleton Papers Inc. of Appleton, Wis. USA.

In addition, the materials for making the aforementioned encapsulates can be obtained from Solutia Inc. (St. Louis, Mo. U.S.A.), Cytec Industries (West Paterson, N.J. U.S.A.), sigma-Aldrich (St. Louis, Mo. U.S.A.), CP Kelco Corp. of San Diego, Calif., USA; BASF AG of Ludwigshafen, Germany; Rhodia Corp. of Cranbury, N.J., USA; Hercules Corp. of Wilmington, Del., USA; Agrium Inc. of Calgary, Alberta, Canada, ISP of N.J. U.S.A., Akzo Nobel of Chicago, Ill., USA; Stroever Shellac Bremen of Bremen, Germany; Dow Chemical Company of Midland, Mich., USA; Bayer AG of Leverkusen, Germany; Sigma-Aldrich Corp., St. Louis, Mo., USA.

Amphiphilic Cleaning Polymers

Preferably, the amphiphilic cleaning polymer is a compound having the following general structure: bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n), wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonated variants thereof.

Amphiphilic alkoxylated grease cleaning polymers of the present invention refer to any alkoxylated polymers having balanced hydrophilic and hydrophobic properties such that they remove grease particles from fabrics and surfaces. Specific embodiments of the amphiphilic alkoxylated grease cleaning polymers of the present invention comprise a core structure and a plurality of alkoxylate groups attached to that core structure.

The core structure may comprise a polyalkylenimine structure comprising, in condensed form, repeating units of formulae (I), (II), (III) and (IV):

wherein # in each case denotes one-half of a bond between a nitrogen atom and the free binding position of a group A¹ of two adjacent repeating units of formulae (I), (II), (III) or (IV); * in each case denotes one-half of a bond to one of the alkoxylate groups; and Al is independently selected from linear or branched C₂-C₆-alkylene; wherein the polyalkylenimine structure consists of 1 repeating unit of formula (I), x repeating units of formula (II), y repeating units of formula (III) and y+1 repeating units of formula (IV), wherein x and y in each case have a value in the range of from 0 to about 150; where the average weight average molecular weight, Mw, of the polyalkylenimine core structure is a value in the range of from about 60 to about 10,000 g/mol.

The core structure may alternatively comprise a polyalkanolamine structure of the condensation products of at least one compound selected from N-(hydroxyalkyl)amines of formulae (I.a) and/or (I.b),

wherein A are independently selected from C₁-C₆-alkylene; R¹, R¹*, R², R²*, R³, R³*, R⁴, R⁴*, R⁵ and R⁵* are independently selected from hydrogen, alkyl, cycloalkyl or aryl, wherein the last three mentioned radicals may be optionally substituted; and R⁶ is selected from hydrogen, alkyl, cycloalkyl or aryl, wherein the last three mentioned radicals may be optionally substituted.

The plurality of alkylenoxy groups attached to the core structure are independently selected from alkylenoxy units of the formula (V)

*A²-O_(m)CH₂—CH₂—O_(n)A³-O_(p)R   (V)

wherein * in each case denotes one-half of a bond to the nitrogen atom of the repeating unit of formula (I), (II) or (IV); A² is in each case independently selected from 1,2-propylene, 1,2-butylene and 1,2-isobutylene; A³ is 1,2-propylene; R is in each case independently selected from hydrogen and C₁-C₄-alkyl; m has an average value in the range of from 0 to about 2; n has an average value in the range of from about 20 to about 50; and p has an average value in the range of from about 10 to about 50.

Specific embodiments of the amphiphilic alkoxylated grease cleaning polymers may be selected from alkoxylated polyalkylenimines having an inner polyethylene oxide block and an outer polypropylene oxide block, the degree of ethoxylation and the degree of propoxylation not going above or below specific limiting values. Specific embodiments of the alkoxylated polyalkylenimines according to the present invention have a minimum ratio of polyethylene blocks to polypropylene blocks (n/p) of about 0.6 and a maximum of about 1.5(x+2y+1)^(1/2). Alkoxylated polyalkyenimines having an n/p ratio of from about 0.8 to about 1.2(x+2y+1)^(1/2) have been found to have especially beneficial properties.

The alkoxylated polyalkylenimines according to the present invention have a backbone which consists of primary, secondary and tertiary amine nitrogen atoms which are attached to one another by alkylene radicals A and are randomly arranged. Primary amino moieties which start or terminate the main chain and the side chains of the polyalkylenimine backbone and whose remaining hydrogen atoms are subsequently replaced by alkylenoxy units are referred to as repeating units of formulae (I) or (IV), respectively. Secondary amino moieties whose remaining hydrogen atom is subsequently replaced by alkylenoxy units are referred to as repeating units of formula (II). Tertiary amino moieties which branch the main chain and the side chains are referred to as repeating units of formula (III).

Since cyclization can occur in the formation of the polyalkylenimine backbone, it is also possible for cyclic amino moieties to be present to a small extent in the backbone. Such polyalkylenimines containing cyclic amino moieties are of course alkoxylated in the same way as those consisting of the noncyclic primary and secondary amino moieties.

The polyalkylenimine backbone consisting of the nitrogen atoms and the groups A¹, has an average molecular weight Mw of from about 60 to about 10,000 g/mole, preferably from about 100 to about 8,000 g/mole and more preferably from about 500 to about 6,000 g/mole.

The sum (x+2y+1) corresponds to the total number of alkylenimine units present in one individual polyalkylenimine backbone and thus is directly related to the molecular weight of the polyalkylenimine backbone. The values given in the specification however relate to the number average of all polyalkylenimines present in the mixture. The sum (x+2y+2) corresponds to the total number amino groups present in one individual polyalkylenimine backbone.

The radicals Al connecting the amino nitrogen atoms may be identical or different, linear or branched C₂-C₆-alkylene radicals, such as 1,2-ethylene, 1,2-propylene, 1,2-butylene, 1,2-isobutylene, 1,2-pentanediyl, 1,2-hexanediyl or hexamethylen. A preferred branched alkylene is 1,2-propylene. Preferred linear alkylene are ethylene and hexamethylene. A more preferred alkylene is 1,2-ethylene.

The hydrogen atoms of the primary and secondary amino groups of the polyalkylenimine backbone are replaced by alkylenoxy units of the formula (V).

*A²-O_(m)CH₂—CH₂—O_(n)A³O_(p)—R   (V)

In this formula, the variables preferably have one of the meanings given below:

A² in each case is selected from 1,2-propylene, 1,2-butylene and 1,2-isobutylene;

preferably A² is 1,2-propylene. A³ is 1,2-propylene; R in each case is selected from hydrogen and C₁-C₄-alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert.-butyl; preferably R is hydrogen. The index m in each case has a value of 0 to about 2; preferably m is 0 or approximately 1; more preferably m is 0. The index n has an average value in the range of from about 20 to about 50, preferably in the range of from about 22 to about 40, and more preferably in the range of from about 24 to about 30. The index p has an average value in the range of from about 10 to about 50, preferably in the range of from about 11 to about 40, and more preferably in the range of from about 12 to about 30.

Preferably the alkylenoxy unit of formula (V) is a non-random sequence of alkoxylate blocks. By non-random sequence it is meant that the [-A²-O—]_(m) is added first (i.e., closest to the bond to the nitrogen atom of the repeating unit of formula (I), (II), or (III)), the [—CH₂—CH₂—O—]_(n) is added second, and the [-A³-O—]_(p) is added third. This orientation provides the alkoxylated polyalkylenimine with an inner polyethylene oxide block and an outer polypropylene oxide block.

The substantial part of these alkylenoxy units of formula (V) is formed by the ethylenoxy units —[CH₂—CH₂—O—]_(n)— and the propylenoxy units —[CH₂—CH₂(CH₃)—O]_(p)—. The alkylenoxy units may additionally also have a small proportion of propylenoxy or butylenoxy units -[A²-O]_(m)—, i.e. the polyalkylenimine backbone saturated with hydrogen atoms may be reacted initially with small amounts of up to about 2 mol, especially from about 0.5 to about 1.5 mol, in particular from about 0.8 to about 1.2 mol, of propylene oxide or butylene oxide per mole of NH— moieties present, i.e. incipiently alkoxylated.

This initial modification of the polyalkylenimine backbone allows, if necessary, the viscosity of the reaction mixture in the alkoxylation to be lowered. However, the modification generally does not influence the performance properties of the alkoxylated polyalkylenimine and therefore does not constitute a preferred measure.

The amphiphilic alkoxylated grease cleaning polymers are present in the fabric and home care products, including but not limited to detergents, of the present invention at levels ranging from about 0.05% to 10% by weight of the fabric and home care product. Embodiments of the fabric and home care products may comprise from about 0.1% to about 5% by weight. More specifically, the embodiments may comprise from about 0.25 to about 2.5% of the grease cleaning polymer.

Non-Immunoequivalent Enzyme and/or Additional Enzymes

The fabric and home care products can comprise one or more enzymes which provide cleaning performance and/or fabric care benefits. Examples of suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ß-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. A typical combination is an enzyme cocktail that may comprise, for example, a protease and lipase in conjunction with amylase. When present in a fabric and home care product, the aforementioned additional enzymes may be present at levels from about 0.00001% to about 2%, from about 0.0001% to about 1% or even from about 0.001% to about 0.5% enzyme protein by weight of the fabric and home care product.

In one aspect preferred enzymes would include a protease. Suitable proteases include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases, such as subtilisins (EC 3.4.21.62). Suitable proteases include those of animal, vegetable or microbial origin. In one aspect, such suitable protease may be of microbial origin. The suitable proteases include chemically or genetically modified mutants of the aforementioned suitable proteases. In one aspect, the suitable protease may be a serine protease, such as an alkaline microbial protease or/and a trypsin-type protease. Examples of suitable neutral or alkaline proteases include:

(a) subtilisins (EC 3.4.21.62), including those derived from Bacillus, such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in U.S. Pat. No. 6,312,936 B1, U.S. Pat. No. 5,679,630, U.S. Pat. No. 4,760,025, U.S. Pat. No. 7,262,042 and WO09/021867.

(b) trypsin-type or chymotrypsin-type proteases, such as trypsin (e.g., of porcine or bovine origin), including the Fusarium protease described in WO 89/06270 and the chymotrypsin proteases derived from Cellumonas described in WO 05/052161 and WO 05/052146.

(c) metalloproteases, including those derived from Bacillus amyloliquefaciens described in WO 07/044993A2.

Preferred proteases include those derived from Bacillus gibsonii or Bacillus Lentus.

Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Savinase®, Primase®, Durazym®, Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®, Ovozyme®, Neutrase®, Everlase® and Esperase® by Novozymes A/S (Denmark), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®, Excellase® and Purafect OXP® by Genencor International, those sold under the tradename Opticlean® and Optimase® by Solvay Enzymes, those available from Henkel/ Kemira, namely BLAP (sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604 with the following mutations S99D+S101 R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R (BLAP with S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP with S3T+V4I+V205I) and BLAP F49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D)—all from Henkel/Kemira; and KAP (Bacillus alkalophilus subtilisin with mutations A230V+S256G+S259N) from Kao.

In one aspect, the fabric and home care product may comprise a protease that is not immunoequivalent to the cold water protease of this invention. For the purposes of this invention, an immunoequivalent protease will have a high degree of identity (>80%) with BPN′ and will cross-react with the same antibody. Suitable non-immunoequivalent enzymes will include those derived from Bacillus Lentus, Bacillus gibsonii and the metalloprotease derived from Bacillus amyloliquefaciens.

Suitable alpha-amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants (variants) are included. A preferred alkaline alpha-amylase is derived from a strain of Bacillus, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or other Bacillus sp., such as Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375 (U.S. Pat. No. 7,153,818) DSM 12368, DSMZ no. 12649, KSM AP1378 (WO 97/00324), KSM K36 or KSM K38 (EP 1,022,334). Preferred amylases include:

(a) the variants described in WO 94/02597, WO 94/18314, WO96/23874 and WO 97/43424, especially the variants with substitutions in one or more of the following positions versus the enzyme listed as SEQ ID No. 2 in WO 96/23874: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

(b) the variants described in U.S. Pat. No. 5,856,164 and WO99/23211, WO 96/23873, WO00/60060 and WO 06/002643, especially the variants with one or more substitutions in the following positions versus the AA560 enzyme listed as SEQ ID No. 12 in WO 06/002643:

26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484, preferably that also contain the deletions of D183* and G184*.

(c) variants exhibiting at least 90% identity with SEQ ID No. 4 in WO06/002643, the wild-type enzyme from Bacillus SP722, especially variants with deletions in the 183 and 184 positions and variants described in WO 00/60060, which is incorporated herein by reference.

(d) variants exhibiting at least 95% identity with the wild-type enzyme from Bacillus sp. 707 (SEQ ID NO:7 in U.S. Pat. No. 6,093, 562), especially those comprising one or more of the he following mutations M202, M208, 5255, R172, and/or M261. Preferably said amylase comprises one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and/or R172Q. Particularly preferred are those comprising the M202L or M202T mutations.

Suitable commercially available alpha-amylases include DURAMYL®, LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®, STAINZYME®, STAINZYME PLUS®, FUNGAMYL® and BAN® (Novozymes A/S, Bagsvaerd, Denmark), KEMZYM® AT 9000 Biozym Biotech Trading GmbH Wehlistrasse 27b A-1200 Wien Austria, RAPIDASE®, PURASTAR®, ENZYSIZE®, OPTISIZE HT PLUS® and PURASTAR OXAM® (Genencor International Inc., Palo Alto, Calif.) and KAM® (Kao, 14-10 Nihonbashi Kayabacho, 1-chome, Chuo-ku Tokyo 103-8210, Japan). In one aspect, suitable amylases include NATALASE®, STAINZYME® and STAINZYME PLUS® and mixtures thereof.

In one aspect, such additional enzyme may be selected from the group consisting of: lipases, including “first cycle lipases” such as those described in U.S. Pat. No. 6,939,702 B1 and US PA 2009/0217464. In one aspect, the lipase is a first-wash lipase, preferably a variant of the wild-type lipase from Thermomyces lanuginosus comprising T231R and N233R mutations. The wild-type sequence is the 269 amino acids (amino acids 23-291) of the Swissprot accession number Swiss-Prot 059952 (derived from Thermomyces lanuginosus (Humicola lanuginosa)). Preferred lipases would include those sold under the tradenames Lipex® and Lipolex®.

In one aspect, other preferred enzymes include microbial-derived endoglucanases exhibiting endo-beta-1,4-glucanase activity (E.C. 3.2.1.4), including a bacterial polypeptide endogenous to a member of the genus Bacillus which has a sequence of at least 90%, 94%, 97% and even 99% identity to the amino acid sequence SEQ ID NO:2 in U.S. Pat. No. 7,141,403B2) and mixtures thereof. Suitable endoglucanases are sold under the tradenames Celluclean® and Whitezyme® (Novozymes A/S, Bagsvaerd, Denmark).

Other preferred enzymes include pectate lyases sold under the tradenames Pectawash®, Pectaway® and mannanases sold under the tradenames Mannaway® (all from Novozymes A/S, Bagsvaerd, Denmark), and Purabrite® (Genencor International Inc., Palo Alto, Calif.).

Adjunct Materials

While not essential for the purposes of the present invention, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the instant fabric and home care products and may be desirably incorporated in certain embodiments of the invention, for example to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the fabric and home care product as is the case with perfumes, colorants, dyes or the like. The levels of any such adjuncts incorporated in any fabric and home care product are in addition to any materials previously recited for incorporation. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the fabric and home care product and the nature of the cleaning operation for which it is to be used. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, perfume delivery systems, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and/or pigments. In addition to the disclosure below, suitable examples of such other adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282, 6,306,812 B1 and 6,326,348 B1 that are incorporated by reference.

As stated, the adjunct ingredients are not essential to Applicants' fabric and home care products. Thus, certain embodiments of Applicants' fabric and home care products do not contain one or more of the following adjuncts materials: surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, perfume delivery systems, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and/or pigments. However, when one or more adjuncts are present, such one or more adjuncts may be present as detailed below:

Perfume Delivery Systems and Perfumes—The compositions of the present invention may comprise a neat perfume and/or perfume technology systems that can be combined to yield the desired scent experience from the store shelf stage of a product, through its total performance cycle. Suitable perfumes include those perfumes that are enduring perfumes and/or quadrant perfumes. Examples of such neat perfumes are disclosed U.S. Pat. No. Nos. 5,500,138; 5,500,154; 6,491,728; 5,500,137 and 5,780,404. Suitable perfume delivery systems, methods of making certain perfume delivery systems and the uses of such perfume delivery systems are disclosed in USPA 2007/0275866 A1. Such perfume delivery systems include:

-   -   Polymer Assisted Delivery (PAD): This perfume delivery         technology uses polymeric materials to deliver perfume         materials. Classical coacervation, water soluble or partly         soluble to insoluble charged or neutral polymers, liquid         crystals, hot melts, hydrogels, perfumed plastics,         microcapsules, nano- and micro-latexes, polymeric film formers,         and polymeric absorbents, polymeric adsorbents, etc. are some         examples. PAD includes but is not limited to:         -   a.) Matrix Systems: The fragrance is dissolved or dispersed             in a polymer matrix or particle. Perfumes, for example, may             be 1) dispersed into the polymer prior to formulating into             the product or 2) added separately from the polymer during             or after formulation of the product. Diffusion of perfume             from the polymer is a common trigger that allows or             increases the rate of perfume release from a polymeric             matrix system that is deposited or applied to the desired             surface (situs), although many other triggers are know that             may control perfume release. Absorption and/or adsorption             into or onto polymeric particles, films, solutions, and the             like are aspects of this technology. Nano- or             micro-particles composed of organic materials (e.g.,             latexes) are examples. Suitable particles include a wide             range of materials including, but not limited to polyacetal,             polyacrylate, polyacrylic, polyacrylonitrile, polyamide,             polyaryletherketone, polybutadiene, polybutylene,             polybutylene terephthalate, polychloroprene, poly ethylene,             polyethylene terephthalate, polycyclohexylene dimethylene             terephthalate, polycarbonate, polychloroprene,             polyhydroxyalkanoate, polyketone, polyester, polyethylene,             polyetherimide, polyethersulfone, polyethylenechlorinates,             polyimide, polyisoprene, polylactic acid, polymethylpentene,             polyphenylene oxide, polyphenylene sulfide, polyphthalamide,             polypropylene, polystyrene, polysulfone, polyvinyl acetate,             polyvinyl chloride, as well as polymers or copolymers based             on acrylonitrile-butadiene, cellulose acetate,             ethylene-vinyl acetate, ethylene vinyl alcohol,             styrene-butadiene, vinyl acetate-ethylene, and mixtures             thereof.         -   “Standard” systems refer to those that are “pre-loaded” with             the intent of keeping the pre-loaded perfume associated with             the polymer until the moment or moments of perfume release.             Such polymers may also suppress the neat product odor and             provide a bloom and/or longevity benefit depending on the             rate of perfume release. One challenge with such systems is             to achieve the ideal balance between 1) in-product stability             (keeping perfume inside carrier until you need it) and 2)             timely release (during use or from dry situs). Achieving             such stability is particularly important during in-product             storage and product aging. This challenge is particularly             apparent for aqueous-based, surfactant-containing products,             such as heavy duty liquid laundry detergents. Many             “Standard” matrix systems available effectively become             “Equilibrium” systems when formulated into aqueous-based             products. One may select an “Equilibrium” system or a             Reservoir system, which has acceptable in-product diffusion             stability and available triggers for release (e.g.,             friction). “Equilibrium” systems are those in which the             perfume and polymer may be added separately to the product,             and the equilibrium interaction between perfume and polymer             leads to a benefit at one or more consumer touch points             (versus a free perfume control that has no polymer-assisted             delivery technology). The polymer may also be pre-loaded             with perfume; however, part or all of the perfume may             diffuse during in-product storage reaching an equilibrium             that includes having desired perfume raw materials (PRMs)             associated with the polymer. The polymer then carries the             perfume to the surface, and release is typically via perfume             diffusion. The use of such equilibrium system polymers has             the potential to decrease the neat product odor intensity of             the neat product (usually more so in the case of pre-loaded             standard system). Deposition of such polymers may serve to             “flatten” the release profile and provide increased             longevity. As indicated above, such longevity would be             achieved by suppressing the initial intensity and may enable             the formulator to use more high impact or low odor detection             threshold (ODT) or low Kovats Index (KI) PRMs to achieve             FMOT benefits without initial intensity that is too strong             or distorted. It is important that perfume release occurs             within the time frame of the application to impact the             desired consumer touch point or touch points. Suitable             micro-particles and micro-latexes as well as methods of             making same may be found in USPA 2005/0003980 A1. Matrix             systems also include hot melt adhesives and perfume             plastics. In addition, hydrophobically modified             polysaccharides may be formulated into the perfumed product             to increase perfume deposition and/or modify perfume             release. All such matrix systems, including for example             polysaccharides and nanolatexes may be combined with other             PDTs, including other PAD systems such as PAD reservoir             systems in the form of a perfume microcapsule (PMC). Polymer             Assisted Delivery (PAD) matrix systems may include those             described in the following references: US Patent             Applications 2004/0110648 A1; 2004/0092414 A1; 2004/0091445             A1 and 2004/0087476 A1; and U.S. Pat. Nos. 6,531,444;             6,024,943; 6,042,792; 6,051,540; 4,540,721 and 4,973,422.         -   Silicones are also examples of polymers that may be used as             PDT, and can provide perfume benefits in a manner similar to             the polymer-assisted delivery “matrix system”. Such a PDT is             referred to as silicone-assisted delivery (SAD). One may             pre-load silicones with perfume, or use them as an             equilibrium system as described for PAD. Suitable silicones             as well as making same may be found in WO 2005/102261; USPA             20050124530A1; USPA 20050143282A1; and WO 2003/015736.             Functionalized silicones may also be used as described in             USPA 2006/003913 A1. Examples of silicones include             polydimethylsiloxane and polyalkyldimethylsiloxanes. Other             examples include those with amine functionality, which may             be used to provide benefits associated with amine-assisted             delivery (AAD) and/or polymer-assisted delivery (PAD) and/or             amine-reaction products (ARP). Other such examples may be             found in U.S. Pat. No. 4,911,852; USPA 2004/0058845 A1; USPA             2004/0092425 A1 and USPA 2005/0003980 A1.         -   b.) Reservoir Systems: Reservoir systems are also known as a             core-shell type technology, or one in which the fragrance is             surrounded by a perfume release controlling membrane, which             may serve as a protective shell. The material inside the             microcapsule is referred to as the core, internal phase, or             fill, whereas the wall is sometimes called a shell, coating,             or membrane. Microparticles or pressure sensitive capsules             or microcapsules are examples of this technology.             Microcapsules of the current invention are formed by a             variety of procedures that include, but are not limited to,             coating, extrusion, spray-drying, interfacial, in-situ and             matrix polymerization. The possible shell materials vary             widely in their stability toward water. Among the most             stable are polyoxymethyleneurea (PMU)-based materials, which             may hold certain PRMs for even long periods of time in             aqueous solution (or product). Such systems include but are             not limited to urea-formaldehyde and/or             melamine-formaldehyde. Gelatin-based microcapsules may be             prepared so that they dissolve quickly or slowly in water,             depending for example on the degree of cross-linking. Many             other capsule wall materials are available and vary in the             degree of perfume diffusion stability observed. Without             wishing to be bound by theory, the rate of release of             perfume from a capsule, for example, once deposited on a             surface is typically in reverse order of in-product perfume             diffusion stability. As such, urea-formaldehyde and             melamine-formaldehyde microcapsules for example, typically             require a release mechanism other than, or in addition to,             diffusion for release, such as mechanical force (e.g.,             friction, pressure, shear stress) that serves to break the             capsule and increase the rate of perfume (fragrance)             release. Other triggers include melting, dissolution,             hydrolysis or other chemical reaction, electromagnetic             radiation, and the like. The use of pre-loaded microcapsules             requires the proper ratio of in-product stability and in-use             and/or on-surface (on-situs) release, as well as proper             selection of PRMs. Microcapsules that are based on             urea-formaldehyde and/or melamine-formaldehyde are             relatively stable, especially in near neutral aqueous-based             solutions. Other suitable microcapsules include             microcapsules having shells that comprise urethane             materials, acrylics and/or vinyl alcohols. These materials             may require a friction trigger which may not be applicable             to all product applications. Other microcapsule materials             (e.g., gelatin) may be unstable in aqueous-based products             and may even provide reduced benefit (versus free perfume             control) when in-product aged. Scratch and sniff             technologies are yet another example of PAD. Perfume             microcapsules (PMC) may include those described in the             following references: US Patent Applications: 2003/0125222             A1; 2003/215417 A1; 2003/216488 A1; 2003/158344 A1;             2003/165692 A1; 2004/071742 A1; 2004/071746 A1; 2004/072719             A1; 2004/072720 A1; 2006/0039934 A1; 2003/203829 A1;             2003/195133 A1; 2004/087477 A1; 2004/0106536 A1; and U.S.             Pat. Nos. 6,645,479 B1; 6,200,949 B1; 4,882,220; 4,917,920;             4,514,461; 6,106,875 and 4,234,627, 3,594,328 and U.S. Pat.             No. RE 32713.

-   Molecule-Assisted Delivery (MAD): Non-polymer materials or molecules     may also serve to improve the delivery of perfume. Without wishing     to be bound by theory, perfume may non-covalently interact with     organic materials, resulting in altered deposition and/or release.     Non-limiting examples of such organic materials include but are not     limited to hydrophobic materials such as organic oils, waxes,     mineral oils, petrolatum, fatty acids or esters, sugars,     surfactants, liposomes and even other perfume raw material (perfume     oils), as well as natural oils, including body and/or other soils.     Perfume fixatives are yet another example. In one aspect,     non-polymeric materials or molecules have a CLogP greater than     about 2. Molecule-Assisted Delivery (MAD) may also include those     described in U.S. Pat. No. 7,119,060 and U.S. Pat. No. 5,506,201.

-   Fiber-Assisted Delivery (FAD): The choice or use of a situs itself     may serve to improve the delivery of perfume. In fact, the situs     itself may be a perfume delivery technology. For example, different     fabric types such as cotton or polyester will have different     properties with respect to ability to attract and/or retain and/or     release perfume. The amount of perfume deposited on or in fibers may     be altered by the choice of fiber, and also by the history or     treatment of the fiber, as well as by any fiber coatings or     treatments. Fibers may be woven and non-woven as well as natural or     synthetic. Natural fibers include those produced by plants, animals,     and geological processes, and include but are not limited to     cellulose materials such as cotton, linen, hemp jute, flax, ramie,     and sisal, and fibers used to manufacture paper and cloth.     Fiber-Assisted Delivery may consist of the use of wood fiber, such     as thermomechanical pulp and bleached or unbleached kraft or sulfite     pulps. Animal fibers consist largely of particular proteins, such as     silk, sinew, catgut and hair (including wool). Polymer fibers based     on synthetic chemicals include but are not limited to polyamide     nylon, PET or PBT polyester, phenol-formaldehyde (PF), polyvinyl     alcohol fiber (PVOH), polyvinyl chloride fiber (PVC), polyolefins     (PP and PE), and acrylic polymers. All such fibers may be pre-loaded     with a perfume, and then added to a product that may or may not     contain free perfume and/or one or more perfume delivery     technologies. In one aspect, the fibers may be added to a product     prior to being loaded with a perfume, and then loaded with a perfume     by adding a perfume that may diffuse into the fiber, to the product.     Without wishing to be bound by theory, the perfume may absorb onto     or be adsorbed into the fiber, for example, during product storage,     and then be released at one or more moments of truth or consumer     touch points.

-   Amine Assisted Delivery (AAD): The amine-assisted delivery     technology approach utilizes materials that contain an amine group     to increase perfume deposition or modify perfume release during     product use. There is no requirement in this approach to pre-complex     or pre-react the perfume raw material(s) and amine prior to addition     to the product. In one aspect, amine-containing AAD materials     suitable for use herein may be non-aromatic; for example,     polyalkylimine, such as polyethyleneimine (PEI), or polyvinylamine     (PVAm), or aromatic, for example, anthranilates. Such materials may     also be polymeric or non-polymeric. In one aspect, such materials     contain at least one primary amine This technology will allow     increased longevity and controlled release also of low ODT perfume     notes (e.g., aldehydes, ketones, enones) via amine functionality,     and delivery of other PRMs, without being bound by theory, via     polymer-assisted delivery for polymeric amines Without technology,     volatile top notes can be lost too quickly, leaving a higher ratio     of middle and base notes to top notes. The use of a polymeric amine     allows higher levels of top notes and other PRMS to be used to     obtain freshness longevity without causing neat product odor to be     more intense than desired, or allows top notes and other PRMs to be     used more efficiently. In one aspect, AAD systems are effective at     delivering PRMs at pH greater than about neutral. Without wishing to     be bound by theory, conditions in which more of the amines of the     AAD system are deprotonated may result in an increased affinity of     the deprotonated amines for PRMs such as aldehydes and ketones,     including unsaturated ketones and enones such as damascone. In     another aspect, polymeric amines are effective at delivering PRMs at     pH less than about neutral. Without wishing to be bound by theory,     conditions in which more of the amines of the AAD system are     protonated may result in a decreased affinity of the protonated     amines for PRMs such as aldehydes and ketones, and a strong affinity     of the polymer framework for a broad range of PRMs. In such an     aspect, polymer-assisted delivery may be delivering more of the     perfume benefit; such systems are a subspecies of AAD and may be     referred to as Amine-Polymer-Assisted Delivery or APAD. In some     cases when the APAD is employed in a composition that has a pH of     less than seven, such APAD systems may also be considered     Polymer-Assisted Delivery (PAD). In yet another aspect, AAD and PAD     systems may interact with other materials, such as anionic     surfactants or polymers to form coacervate and/or coacervates-like     systems. In another aspect, a material that contains a heteroatom     other than nitrogen, for example sulfur, phosphorus or selenium, may     be used as an alternative to amine compounds. In yet another aspect,     the aforementioned alternative compounds can be used in combination     with amine compounds. In yet another aspect, a single molecule may     comprise an amine moiety and one or more of the alternative     heteroatom moieties, for example, thiols, phosphines and selenols.     Suitable AAD systems as well as methods of making same may be found     in US Patent Applications 2005/0003980 A1; 2003/0199422 A1;     2003/0036489 A1; 2004/0220074 A1 and U.S. Pat. No. 6,103,678.

-   Cyclodextrin Delivery System (CD): This technology approach uses a     cyclic oligosaccharide or cyclodextrin to improve the delivery of     perfume. Typically a perfume and cyclodextrin (CD) complex is     formed. Such complexes may be preformed, formed in-situ, or formed     on or in the situs. Without wishing to be bound by theory, loss of     water may serve to shift the equilibrium toward the CD-Perfume     complex, especially if other adjunct ingredients (e.g., surfactant)     are not present at high concentration to compete with the perfume     for the cyclodextrin cavity. A bloom benefit may be achieved if     water exposure or an increase in moisture content occurs at a later     time point. In addition, cyclodextrin allows the perfume formulator     increased flexibility in selection of PRMs. Cyclodextrin may be     pre-loaded with perfume or added separately from perfume to obtain     the desired perfume stability, deposition or release benefit.     Suitable CDs as well as methods of making same may be found in USPA     2005/0003980 A1 and 2006/0263313 A1 and U.S. Pat. Nos. 5,552,378;     3,812,011; 4,317,881; 4,418,144 and 4,378,923.

-   Starch Encapsulated Accord (SEA): The use of a starch encapsulated     accord (SEA) technology allows one to modify the properties of the     perfume, for example, by converting a liquid perfume into a solid by     adding ingredients such as starch. The benefit includes increased     perfume retention during product storage, especially under     non-aqueous conditions. Upon exposure to moisture, a perfume bloom     may be triggered. Benefits at other moments of truth may also be     achieved because the starch allows the product formulator to select     PRMs or PRM concentrations that normally cannot be used without the     presence of SEA. Another technology example includes the use of     other organic and inorganic materials, such as silica to convert     perfume from liquid to solid. Suitable SEAs as well as methods of     making same may be found in USPA 2005/0003980 A1 and U.S. Pat. No.     6,458,754 B1.

In one aspect, SEA's may be made by preparing a mixture comprising starch, water, acid and a perfume, the acid being incorporated in the mixture in an amount sufficient to lower the pH of the starch-water mixture by at least 0.25 units; and atomising and drying the mixture thereby forming encapsulated perfume. In the first step in the process of perfume encapsulation, an aqueous mixture is prepared comprising starch, water, perfume and acid. These ingredients may be added in any order, but usually the starch-water mixture is prepared first and subsequently, either sequentially or together, the acid and perfume are added. When they are added sequentially, the acid may be added prior to the ingredient for encapsulation. Alternatively, the acid is added after the ingredient for encapsulation. The concentration of starch in the aqueous mixture may be from as low as 5 or 10 wt % to as high as 60 or even 75 wt %. Generally the concentration of starch in the mixture is from 20 to 50 wt %, more usually around 25 to 40 wt % in the aqueous mixture.

-   Suitable starches can be made from raw starch, pregelatinized     starch, modified starch derived from tubers, legumes, cereal and     grains for example corn starch, wheat starch, rice starch, waxy corn     starch, oat starch, cassava starch, waxy barley starch, waxy rice     starch, sweet rice starch, amioca, potato starch, tapioca starch and     mixtures thereof. Modified starches may be particularly suitable for     use in the present invention, and these include hydrolyzed starch,     acid thinned starch, starch having hydrophobic groups, such as     starch esters of long chain hydrocarbons (C5 or greater), starch     acetates, starch octenyl succinate and mixtures thereof. In one     aspect, starch esters, such as starch octenyl succinates are     employed. -   The term “hydrolyzed starch” refers to oligosaccharide-type     materials that are typically obtained by acid and/or enzymatic     hydrolysis of starches, preferably corn starch. It may be preferred     to include in the starch water-mixture, a starch ester. Particularly     preferred are the modified starches comprising a starch derivative     containing a hydrophobic group or both a hydrophobic and a     hydrophilic group which has been degraded by at least one enzyme     capable of cleaving the 1,4 linkages of the starch molecule from the     non-reducing ends to produce short chained saccharides to provide     high oxidation resistance while maintaining substantially high     molecular weight portions of the starch base. The aqueous starch     mixture may also include a plasticizer for the starch. Suitable     examples include monosaccharides, disaccharides, oligosaccharides     and maltodextrins, such as glucose, sucrose, sorbitol, gum arabic,     guar gums and maltodextrin. -   The acid used in the process of the invention may be any acid.     Examples include sulphuric acid, nitric acid, hydrochloric acid,     sulphamic acid and phosphonic acid. In one aspect, carboxylic     organic acids are employed. In another aspect, organic acids     comprising more than one carboxylic acid groups are employed.     Examples of suitable organic acids include citric acid, tartaric     acid, maleic acid, malic acid, succinic acid, sebacic acid, adipic     acid, itaconic acid, acetic acid and ascorbic acid, etc. In one     aspect, saturated acids, such as citric acid, are employed. -   Suitable perfumes for encapsulation include the HIA perfumes     including those having a boiling point determined at the normal     standard pressure of about 760 mmHg of 275° C. or lower, an     octanol/water partition coefficient P of about 2000 or higher and an     odour detection threshold of less than or equal 50 parts per billion     (ppb). In one aspect, the perfume may have log P of 2 or higher.     Suitable perfumes may be selected from the group consisting of     3-(4-t-butylphenyl)-2-methyl propanal, 3-(4-t-butylphenyl)-propanal,     3-(4-isopropylphenyl)-2-methylpropanal,     methylenedioxyphenyl)-2-methylpropanal, and 2,6-dimethyl-5-heptenal,     Alpha-damascone, Delta-damascone, Iso-damascone, Beta-damascenone,     6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone,     methyl-7,3-dihydro-2H-1,5-benzodioxepine-3-one,     2-[2-(4-methyl-3-cyclohexenyl-1-yl)propyl]cyclopentan-2-one,     2-sec-butylcyclohexanone, and Alpha-dihydro ionone, linalool,     ethyllinalool, tetrahydrolinalool, and dihydromyrcenol. -   Suitable ingredients can be obtained from Givaudan of Mount Olive,     N.J., USA, International Flavors & Fragrances of South Brunswick,     N.J., USA, or Quest of Naarden, Netherlands. -   Following the formation of the aqueous mixture comprising starch,     water, perfumes and acid, the mixture is mixed under high shear to     form an emulsion or dispersion of ingredient for encapsulation in     the aqueous starch solution. -   Any suitable technique may then be used for the final stage of     processing where the aqueous mixture including acid and perfumes is     atomised and dried. Suitable techniques include, but are not limited     to those known in the art including spray drying, extrusion, spray     chilling/crystallisation methods, fluid bed coating and the use of     phase transfer catalysts to promote interfacial polymerization.     Spray efficiencies may be increased by methods known in the art,     such as by using high drying towers, lightly oiling the chamber     walls, or using preconditioned air in which the moisture has been     substantially removed. -   Inorganic Carrier Delivery System (ZIC): This technology relates to     the use of porous zeolites or other inorganic materials to deliver     perfumes. Perfume-loaded zeolite may be used with or without adjunct     ingredients used for example to coat the perfume-loaded zeolite     (PLZ) to change its perfume release properties during product     storage or during use or from the dry situs. Suitable zeolite and     inorganic carriers as well as methods of making same may be found in     USPA 2005/0003980 A1 and U.S. Pat. Nos. 5,858,959; 6,245,732 B1;     6,048,830 and 4,539,135. Silica is another form of ZIC. Another     example of a suitable inorganic carrier includes inorganic tubules,     where the perfume or other active material is contained within the     lumen of the nano- or micro-tubules. Preferably, the perfume-loaded     inorganic tubule (or Perfume-Loaded Tubule or PLT) is a mineral     nano- or micro-tubule, such as halloysite or mixtures of halloysite     with other inorganic materials, including other clays. The PLT     technology may also comprise additional ingredients on the inside     and/or outside of the tubule for the purpose of improving in-product     diffusion stability, deposition on the desired situs or for     controlling the release rate of the loaded perfume. Monomeric and/or     polymeric materials, including starch encapsulation, may be used to     coat, plug, cap, or otherwise encapsulate the PLT. Suitable PLT     systems as well as methods of making same may be found in U.S. Pat.     No. 5,651,976. -   Pro-Perfume (PP): This technology refers to perfume technologies     that result from the reaction of perfume materials with other     substrates or chemicals to form materials that have a covalent bond     between one or more PRMs and one or more carriers. The PRM is     converted into a new material called a pro-PRM (i.e., pro-perfume),     which then may release the original PRM upon exposure to a trigger     such as water or light. Pro-perfumes may provide enhanced perfume     delivery properties such as increased perfume deposition, longevity,     stability, retention, and the like. Pro-perfumes include those that     are monomeric (non-polymeric) or polymeric, and may be pre-formed or     may be formed in-situ under equilibrium conditions, such as those     that may be present during in-product storage or on the wet or dry     situs. Nonlimiting examples of pro-perfumes include Michael adducts     (e.g., beta-amino ketones), aromatic or non-aromatic imines (Schiffs     Bases), oxazolidines, beta-keto esters, and orthoesters. Another     aspect includes compounds comprising one or more beta-oxy or     beta-thio carbonyl moieties capable of releasing a PRM, for example,     an alpha, beta-unsaturated ketone, aldehyde or carboxylic ester. The     typical trigger for perfume release is exposure to water; although     other triggers may include enzymes, heat, light, pH change,     autoxidation, a shift of equilibrium, change in concentration or     ionic strength and others. For aqueous-based products,     light-triggered pro-perfumes are particularly suited. Such     photo-pro-perfumes (PPPs) include but are not limited to those that     release coumarin derivatives and perfumes and/or pro-perfumes upon     being triggered. The released pro-perfume may release one or more     PRMs by means of any of the above mentioned triggers. In one aspect,     the photo-pro-perfume releases a nitrogen-based pro-perfume when     exposed to a light and/or moisture trigger. In another aspect, the     nitrogen-based pro-perfume, released from the photo-pro-perfume,     releases one or more PRMs selected, for example, from aldehydes,     ketones (including enones) and alcohols. In still another aspect,     the PPP releases a dihydroxy coumarin derivative. The     light-triggered pro-perfume may also be an ester that releases a     coumarin derivative and a perfume alcohol. In one aspect the     pro-perfume is a dimethoxybenzoin derivative as described in USPA     2006/0020459 A1. In another aspect the pro-perfume is a 3′,     5′-dimethoxybenzoin (DMB) derivative that releases an alcohol upon     exposure to electromagnetic radiation. In yet another aspect, the     pro-perfume releases one or more low ODT PRMs, including tertiary     alcohols such as linalool, tetrahydrolinalool, or dihydromyrcenol.     Suitable pro-perfumes and methods of making same can be found in     U.S. Pat. Nos. 7,018,978 B2; 6,987,084 B2; 6,956,013 B2; 6,861,402     B1; 6,544,945 B1; 6,093,691; 6,277,796 B1; 6,165,953; 6,316,397 B1;     6,437,150 B1; 6,479,682 B1; 6,096,918; 6,218,355 B1; 6,133,228;     6,147,037; 7,109,153 B2; 7,071,151 B2; 6,987,084 B2; 6,610,646 B2     and 5,958,870, as well as can be found in USPA 2005/0003980 A1 and     USPA 2006/0223726 A1.     -   a.) Amine Reaction Product (ARP): For purposes of the present         application, ARP is a subclass or species of PP. One may also         use “reactive” polymeric amines in which the amine functionality         is pre-reacted with one or more PRMs to form an amine reaction         product (ARP). Typically the reactive amines are primary and/or         secondary amines, and may be part of a polymer or a monomer         (non-polymer). Such ARPs may also be mixed with additional PRMs         to provide benefits of polymer-assisted delivery and/or         amine-assisted delivery. Nonlimiting examples of polymeric         amines include polymers based on polyalkylimines, such as         polyethyleneimine (PEI), or polyvinylamine (PVAm). Nonlimiting         examples of monomeric (non-polymeric) amines include hydroxyl         amines, such as 2-aminoethanol and its alkyl substituted         derivatives, and aromatic amines such as anthranilates. The ARPs         may be premixed with perfume or added separately in leave-on or         rinse-off applications. In another aspect, a material that         contains a heteroatom other than nitrogen, for example oxygen,         sulfur, phosphorus or selenium, may be used as an alternative to         amine compounds. In yet another aspect, the aforementioned         alternative compounds can be used in combination with amine         compounds. In yet another aspect, a single molecule may comprise         an amine moiety and one or more of the alternative heteroatom         moieties, for example, thiols, phosphines and selenols. The         benefit may include improved delivery of perfume as well as         controlled perfume release. Suitable ARPs as well as methods of         making same can be found in USPA 2005/0003980 A1 and U.S. Pat.         No. 6,413,920 B1.

In one aspect, the amine reaction product's perfume component, which is reacted with the amine to form the amine reaction product, is selected from a perfume comprising a ketone moiety and/or an aldehyde moiety. In one aspect, such perfumes comprise a chain containing at least 5 carbon atoms. In one aspect, suitable perfumes comprising a ketone moiety may be selected from Alpha Damascone, Delta Damascone, Iso Damascone, Carvone, Gamma-Methyl-Ionone, Iso-E-Super, 2,4,4,7-Tetramethyl-oct-6-en-3-one, Benzyl Acetone, Beta Damascone, Damascenone, methyl dihydrojasmonate, methyl cedrylone, and mixtures thereof. In one aspect, suitable perfumes comprising an aldehyde moiety may be selected from 1-decanal, benzaldehyde, florhydral, 2,4-dimethyl-3-cyclohexen-1-carboxaldehyde; cis/trans-3,7-dimethyl-2,6-octadien-1-al; heliotropin; 2,4,6-trimethyl-3-cyclohexene-1-carboxaldehyde; 2,6-nonadienal; alpha-n-amyl cinnamic aldehyde, alpha-n-hexyl cinnamic aldehyde, P.T. Bucinal, lyral, cymal, methyl nonyl acetaldehyde, hexanal, trans-2-hexenal, and mixture thereof. In one aspect, the suitable perfume mat be selected from undecylenic aldehyde, undecalactone gamma, heliotropin, dodecalactone gamma, p-anisic aldehyde, para hydroxy-phenyl-butanone, cymal, benzyl acetone, ionone alpha, p.t.bucinal, damascenone, ionone beta and methyl-nonyl ketone, and/or mixtures thereof. Typically the level of perfume may be from 10% to 90%, from 30% to 85%, or even from 45% to 80% by weight of the amine reaction product. In one aspect, suitable amine reaction products are those resulting from the reaction of polethyleneimine polymer like Lupasol polymers, with one or more of the following Alpha Damascone, Delta Damascone, Carvone, Hedione, Florhydral, Lilial, Heliotropine, Gamma-Methyl-Ionone and 2,4-dimethyl-3-cyclohexen-1-carboxaldehyde; amine reaction products are those resulting from the reaction of Astramol Dendrimers with Carvone and amine reaction products resulting from the reaction of ethyl-4-amino benzoate with 2,4-dimethyl-3-cyclohexen-l-carboxaldehyde. In one aspect, suitable amine reaction products are those resulting from the reaction of Lupasol HF with Delta Damascone; LupasolG35 with Alpha Damascone; LupasolG100 with 2,4-dimethyl-3-cyclohexen-1-carboxaldehyde, ethyl-4-amino benzoate with 2,4-dimethyl-3-cyclohexen-1-carboxaldehyde.

-   In one aspect, suitable primary and/or secondary amine containing     compounds are characterized by an Odor Intensity Index of less than     that of a 1% solution of methylanthranilate in dipropylene glycol. -   A general structure for a suitable primary amine compound is as     follows:

B—(NH₂)_(n)

wherein B is a carrier material, and n is an index of value of at least 1.

-   Suitable compounds comprising a secondary amine group may have a     structure similar to the above excepted that the compound comprises     one or more —NH— moieties in addition to any —NH2 moieties. Thus,     such an amine compound may have the formula:

B—(NH₂)_(n); B—(NH)_(n); B—(NH)_(n)—(NH₂)_(n)

wherein B is a carrier material, and each n is independently an index of value of at least 1.

-   In one aspect, B carriers may be inorganic having non-or     substantially non carbon based backbones, or organic carriers having     essentially carbon bond backbones. -   Suitable inorganic carriers include mono or polymers or     organic-organosilicon copolymers of amino derivatised organo silane,     siloxane, silazane, alumane, aluminum siloxane, or aluminum silicate     compounds. Typical examples of such carriers are: organosiloxanes     with at least one primary amine moiety like the diaminoalkylsiloxane     [H₂NCH₂(CH₃)₂Si]O, or the organoaminosilane (C₆H₅) 3SiNH₂ (described     in: Chemistry and Technology of Silicone, W. Noll, Academic Press     Inc. 1998, London, pp 209, 106). Mono or polymer or     organic-organosilicon copolymers containing one or more     organosilylhydrasine moiety are also suitable. A typical example of     such a carrier material is N,N′-bis(trimethylsilyl)hydrazine     (Me₃Si)₂NNH₂. Typical suitable amines comprising an organic carrier     include aminoaryl derivatives, polyamines, aminoacids and     derivatives, substituted amines and amides, glucamines, dendrimers     and amino-substituted mono-, di-, oligo-, poly-saccharides. -   The amine compound may be interrupted or substituted by linkers or     cellulose substantive group. A general formula for this amine     compound is as follows:

NH_(2n)-L_(m)-B-L_(m)-R*_(m);

wherein each m is an index of value 0 or at least 1, and n is an index of value of at least 1 as defined herein before. As can be seen above, the amine group is linked to a carrier molecule as defined by classes hereinafter described. The primary and/or secondary amine group is either directly linked to the carrier group or via a linker group L. The carrier can also be substituted by a R* substituent, and R* can be linked to the carrier either directly or via a linker group L. R* can also contain branching groups like e.g. tertiary amine and amide groups.

-   It is important for the purpose of the invention that the amine     compound comprises at least one primary and/or secondary amine group     to react with the perfume aldehyde and/or ketone to form the     reaction products. Such reaction is typically known as a Schiff base     reaction as a Schiff base is formed. The amine compound is not     limited to having only one amine function. Indeed, more preferably,     the amine compound comprises more than one amine function, thereby     enabling the amine compound to react with several aldehydes and/or     ketones. Accordingly, reaction products carrying mixed aldehyde(s)     and/or ketone(s) can be achieved, thereby resulting in a mixed     release of such fragrances.

Bleaching Agents—The fabric and home care products of the present invention may comprise one or more bleaching agents. Suitable bleaching agents other than bleaching catalysts include photobleaches, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, pre-formed peracids and mixtures thereof. In general, when a bleaching agent is used, the fabric and home care products of the present invention may comprise from about 0.1% to about 50% or even from about 0.1% to about 25% bleaching agent by weight of the subject fabric and home care product. Examples of suitable bleaching agents include:

(1) photobleaches for example sulfonated zinc phthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes and mixtures thereof;

(2) preformed peracids: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone®, and mixtures thereof. Suitable percarboxylic acids include hydrophobic and hydrophilic peracids having the formula R—(C═O)O—O-M wherein R is an alkyl group, optionally branched, having, when the peracid is hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when the peracid is hydrophilic, less than 6 carbon atoms or even less than 4 carbon atoms; and M is a counterion, for example, sodium, potassium or hydrogen;

(3) sources of hydrogen peroxide, for example, inorganic perhydrate salts, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulphate, perphosphate, persilicate salts and mixtures thereof. In one aspect of the invention the inorganic perhydrate salts are selected from the group consisting of sodium salts of perborate, percarbonate and mixtures thereof. When employed, inorganic perhydrate salts are typically present in amounts of from 0.05 to 40 wt %, or 1 to 30 wt % of the overall fabric and home care product and are typically incorporated into such fabric and home care products as a crystalline solid that may be coated. Suitable coatings include, inorganic salts such as alkali metal silicate, carbonate or borate salts or mixtures thereof, or organic materials such as water-soluble or dispersible polymers, waxes, oils or fatty soaps; and

(4) bleach activators having R—(C═O)-L wherein R is an alkyl group, optionally branched, having, when the bleach activator is hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when the bleach activator is hydrophilic, less than 6 carbon atoms or even less than 4 carbon atoms; and L is leaving group. Examples of suitable leaving groups are benzoic acid and derivatives thereof—especially benzene sulphonate. Suitable bleach activators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzene sulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED) and nonanoyloxybenzene sulphonate (NOBS). Suitable bleach activators are also disclosed in WO 98/17767. While any suitable bleach activator may be employed, in one aspect of the invention the subject fabric and home care product may comprise NOBS, TAED or mixtures thereof.

When present, the peracid and/or bleach activator is generally present in the fabric and home care product in an amount of from about 0.1 to about 60 wt %, from about 0.5 to about 40 wt % or even from about 0.6 to about 10 wt % based on the fabric and home care product. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activator may be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

Surfactants—The fabric and home care products according to the present invention may comprise a surfactant or surfactant system wherein the surfactant can be selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof. When present, surfactant is typically present at a level of from about 0.1% to about 60%, from about 1% to about 50% or even from about 5% to about 40% by weight of the subject fabric and home care product.

Builders—The fabric and home care products of the present invention may comprise one or more detergent builders or builder systems. When a builder is used, the subject fabric and home care product will typically comprise at least about 1%, from about 5% to about 60% or even from about 10% to about 40% builder by weight of the subject fabric and home care product.

Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicate builders and polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof.

Chelating Agents—The fabric and home care products herein may contain a chelating agent. Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof. When a chelating agent is used, the subject fabric and home care product may comprise from about 0.005% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject fabric and home care product. Suitable chelants include DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid), DTPMP (Diethylene triamine penta(methylene phosphonic acid)), 1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylene triamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetri-acetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and derivatives thereof.

Dye Transfer Inhibiting Agents—The fabric and home care products of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject fabric and home care product, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the fabric and home care product.

Brighteners—The fabric and home care products of the present invention can also contain additional components that may tint articles being cleaned, such as fluorescent brighteners. Suitable fluorescent brightener levels include lower levels of from about 0.01, from about 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt %.

Dispersants—The fabric and home care products of the present invention can also contain dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

Enzyme Stabilizers—Enzymes for use in detergents can be stabilized by various techniques. The enzymes employed herein can be stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the finished fabric and home care products that provide such ions to the enzymes. In case of aqueous fabric and home care products comprising protease, a reversible protease inhibitor, such as a boron compound, or compounds such as calcium formate, sodium formate and 1,2-propane diol can be added to further improve stability.

Catalytic Metal Complexes—Applicants' compositions may include catalytic metal complexes. One type of metal-containing bleach catalyst is a catalyst system comprising a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, an auxiliary metal cation having little or no bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and water-soluble salts thereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.

-   If desired, the compositions herein can be catalyzed by means of a     manganese compound. Such compounds and levels of use are well known     in the art and include, for example, the manganese-based catalysts     disclosed in U.S. Pat. No. 5,576,282. -   Cobalt bleach catalysts useful herein are known, and are described,     for example, in U.S. Pat. No. 5,597,936; U.S. Pat. No. 5,595,967.     Such cobalt catalysts are readily prepared by known procedures, such     as taught for example in U.S. Pat. No. 5,597,936, and U.S. Pat. No.     5,595,967. -   Compositions herein may also suitably include a transition metal     complex of ligands such as bispidones (WO 05/042532 A1) and/or     macropolycyclic rigid ligands—abbreviated as “MRLs”. As a practical     matter, and not by way of limitation, the compositions and processes     herein can be adjusted to provide on the order of at least one part     per hundred million of the active MRL species in the aqueous washing     medium, and will typically provide from about 0.005 ppm to about 25     ppm, from about 0.05 ppm to about 10 ppm, or even from about 0.1 ppm     to about 5 ppm, of the MRL in the wash liquor. -   Suitable transition-metals in the instant transition-metal bleach     catalyst include, for example, manganese, iron and chromium.     Suitable MRLs include     5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. -   Suitable transition metal MRLs are readily prepared by known     procedures, such as taught for example in WO 00/32601, and U.S. Pat.     No. 6,225,464. -   Solvents—Suitable solvents include water and other solvents such as     lipophilic fluids. Examples of suitable lipophilic fluids include     siloxanes, other silicones, hydrocarbons, glycol ethers, glycerine     derivatives such as glycerine ethers, perfluorinated amines,     perfluorinated and hydrofluoroether solvents, low-volatility     nonfluorinated organic solvents, diol solvents, other     environmentally-friendly solvents and mixtures thereof.

Processes of Making Fabric and Home Care Products

The fabric and home care products of the present invention can be formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in Applicants' examples and in U.S. Pat. No. 4,990,280; U.S. 20030087791A1; U.S. 20030087790A1; U.S. 20050003983A1; U.S. 20040048764A1; U.S. Pat. No. 4,762,636; U.S. Pat. No. 6,291,412; U.S. 20050227891A1; EP 1070115A2; U.S. Pat. No. 5,879,584; U.S. Pat. No. 5,691,297; U.S. Pat. No. 5,574,005; U.S. Pat. No. 5,569,645; U.S. Pat. No. 5,565,422; U.S. Pat. No. 5,516,448; U.S. Pat. No. 5,489,392; U.S. Pat. No. 5,486,303 all of which are incorporated herein by reference.

Method of Use

The present invention includes a method for cleaning and/or treating a situs inter alia a surface or fabric. In one aspect, such method comprises the steps of optionally washing and/or rinsing said surface or fabric, contacting said surface or fabric with any fabric and home care product disclosed in this specification then optionally washing and/or rinsing said surface or fabric is disclosed.

As used herein, washing includes but is not limited to, scrubbing, and mechanical agitation. Drying of such surfaces or fabrics may be accomplished by any one of the common means employed either in domestic or industrial settings. Such means include but are not limited to forced air or still air drying at ambient or elevated temperatures at pressures between 5 and 0.01 atmospheres in the presence or absence of electromagnetic radiation, including sunlight, infrared, ultraviolet and microwave irradiation. In one aspect, said drying may be accomplished at temperatures above ambient by employing an iron wherein, for example, said fabric may be in direct contact with said iron for relatively short or even extended periods of time and wherein pressure may be exerted beyond that otherwise normally present due to gravitational force. In another aspect, said drying may be accomplished at temperatures above ambient by employing a dryer. Apparatus for drying fabric is well known and it is frequently referred to as a clothes dryer. In addition to clothes such appliances are used to dry many other items including towels, sheets, pillowcases, diapers and so forth and such equipment has been accepted as a standard convenience in many nations of the world substantially replacing the use of clothes lines for drying of fabric. Most dryers in use today use heated air which is passed over and or through the fabric as it is tumbled within the dryer. The air may be heated, for example, either electronically, via gas flame, or even with microwave radiation. Such air may be heated from about 15° C. to about 400° C., from about 25° C. to about 200° C., from about 35° C. to about 100° C., or even from about 40° C. to about 85° C. and used in the dryer to dry a surface and/or a fabric. As will be appreciated by one skilled in the art, the cleaning compositions of the present invention are ideally suited for use in laundry applications. Accordingly, the present invention includes a method for laundering a fabric. The method comprises the steps of contacting a fabric to be laundered with a said cleaning laundry solution comprising at least one embodiment of Applicants' cleaning composition, cleaning additive or mixture thereof. The fabric may comprise most any fabric capable of being laundered in normal consumer or institutional use conditions. The solution preferably has a pH of from about 8 to about 10.5. The compositions may be employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. The water temperatures typically range from about 5° C. to about 90° C. The water to fabric ratio is typically from about 1:1 to about 30:1.

Test Methods

-   Test Method 1

A protocol to define whether a dye or pigment material is a fabric hueing agent for the purpose of the invention is given here:

-   1.) Fill two tergotometer pots with 800 ml of Newcastle upon Tyne,     UK, City Water (˜12 grains per US gallon total hardness, supplied by     Northumbrian Water, Pity Me, Durham, Co. Durham, UK). -   2) Insert pots into tergotometer, with water temperature controlled     at 30° C. and agitation set at 40 rpm for the duration of the     experiment. -   3) Add 4.8 g of IEC-B detergent (IEC 60456 Washing Machine Reference     Base Detergent Type B), supplied by wfk, Brüggen-Bracht, Germany, to     each pot. -   4) After two minutes, add 2.0 mg active colorant to the first pot. -   5) After one minute, add 50 g of flat cotton vest (supplied by     Warwick Equest, Consett, County Durham, UK), cut into 5cm x 5cm     swatches, to each pot. -   6) After 10 minutes, drain the pots and re-fill with cold Water (16°     C.) having a water hardness of 14.4 English Clark Degrees Hardness     with a 3:1 Calcium to Magnesium molar ratio. -   7) After 2 minutes rinsing, remove fabrics. -   8) Repeat steps 3-7 for a further three cycles using the same     treatments. -   9) Collect and line dry the fabrics indoors for 12 hours. -   10) Analyse the swatches using a Hunter Miniscan spectrometer fitted     with D65 illuminant and UVA cutting filter, to obtain Hunter a     (red-green axis) and Hunter b (yellow-blue axis) values. -   11) Average the Hunter a and Hunter b values for each set of     fabrics. If the fabrics treated with colorant under assessment show     an average difference in hue of greater than 0.2 units on either the     a axis or b axis, it is deemed to be a fabric hueing agent for the     purpose of the invention. -   Test Method 2—For Test Method 2, the BMI microswatch assay is run     using the detergent compositions 1, 2 or 4 from Table 1-3. The     detergent is dissolved in water that has a hardness of 6 gpg and     adjusted to a temperature of 16° C. Performance of the variant     enzymes is then determined as per the BMI microswatch assay     described. The performance index is determined by comparing the     performance of the variant with that of the enzyme of SEQ ID NO:2,     SEQ ID NO:4 or SEQ ID NO:6, with in all cases the enzyme dosage     being 1 ppm. This Method is described in further detail in Example     31. -   Test Method 3—For Test Method 3 the BMI microswatch assay is run     using the detergent compositions 1, 2 or 4 from Table 1-3. The     detergent is dissolved in water that has a hardness of 6 gpg and     adjusted to a temperature of 16° C. Performance of the variant     enzymes is then determined as per the BMI microswatch assay     described. The performance index is determined by comparing the     performance of the variant with that of the enzyme of SEQ ID NO:2,     SEQ ID NO:4 or SEQ ID NO:6, with in all cases the enzyme dosage     being 0.4 ppm. This Method is described in further detail in Example     1. -   Test Method 4—For Test Method 4, the BMI microswatch assay is run     using the detergent composition 10 from Table 19-4. The detergent is     dissolved in water that has a hardness of 12 gpg and adjusted to a     temperature of 16° C. Performance of the variant enzymes is then     determined as per the BMI microswatch assay described. The     performance index is determined by comparing the performance of the     variant with that of the enzyme of SEQ ID NO:755, with in all cases     the enzyme dosage being 1.6 ppm. Enzymes having a performance index     of 1.1 or greater are viewed to be Series 1 GG36 cold water     proteases. This Method is described in further detail in Example 19. -   Test Method 5—For Test Method 5, the BMI microswatch assay is run     using the detergent composition 7 from Table 19-4. The detergent is     dissolved in water that has a hardness of 6 gpg and adjusted to a     temperature of 16° C. Performance of the variant enzymes is then     determined as per the BMI microswatch assay described. The     performance index is determined by comparing the performance of the     variant with that of the enzyme of SEQ ID NO:755, with in all cases     the enzyme dosage being 4 ppm. Enzymes having a performance index of     1.1 or greater are viewed to be Series 1 GG36 cold water proteases.     This Method is described in further detail in Example 19. -   Test Method 6—For Test Method 6 the BMI microswatch assay is run     using the detergent composition 7 from Table 19-4. The detergent is     dissolved in water that has a hardness of 6 gpg and adjusted to a     temperature of 16° C. Performance of the variant enzymes is then     determined as per the BMI microswatch assay described. The     performance index is determined by comparing the performance of the     variant with that of a reference enzyme, said reference enzyme being     the enzyme of SEQ ID NO:755 consisting the A158E mutation, with in     all cases the enzyme dosage being 4 ppm. Enzymes having a     performance index of 1.0 or greater are viewed to be Series 1

GG36 cold water proteases. This Method is described in further detail in Example 19.

-   Test Method 7—Electrical conductivity of an aqueous solution is     assayed according to the standard method ASTM D1125 and reported in     units of milliSiemens/cm, abbreviated to mS/cm in this patent.

Experimental

In the experimental disclosure which follow, the following abbreviations apply: PI (Performance Index), ppm (parts per million); M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); rpm (revolutions per minute); GH (degrees German hardness); H₂O (water); dH₂O (deionized water); HCl (hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl (sodium chloride); w/v (weight to volume); v/v (volume to volume); w/w (weight to weight); g (gravity); OD (optical density); ppm (parts per million); Dulbecco's phosphate buffered solution (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto-Tryptone, 24 g/l glycerol, 2.31 g/l KH₂PO₄, and 12.54 g/l K₂HPO₄); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at 600 nm); A₄₀₅ (absorbance at 405 nm); Vmax (the maximum initial velocity of an enzyme catalyzed reaction); PAGE (polyacrylamide gel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST (PBS+0.25% TWEEN®-20); PEG (polyethylene glycol); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPES buffered saline); Tris-HCl (tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine (N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino) ethane-sulfonic acid); TAPS (3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS (3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide); DTT (1,4-dithio-DL-threitol); SA (sinapinic acid (s,5-dimethoxy-4-hydroxy cinnamic acid); TCA (trichloroacetic acid); Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP (Tris[2-carboxyethyl] phosphine); Ci (Curies); mCi (milliCuries); μCi (microCuries); HPLC (high-performance liquid chromatography); RP-HPLC (reverse phase high pressure liquid chromatography); TLC (thin layer chromatography); MALDI-TOF (matrix-assisted laser desorption/ionization—time of flight); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Thermus aquaticus DNA polymerase); Klenow (DNA polymerase I large (Klenow) fragment); EGTA (ethylene glycol-bis(ß-aminoethyl ether) N,N,N′,N′-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla (β-lactamase or ampicillin-resistance gene); HDL (heavy duty liquid); HDD (heavy duty powder detergent); HSG (high suds granular detergent); CEE (Central and Eastern Europe); WE (Western Europe); NA, when used in reference to detergents (North America); Japan and JPN, when used in reference to detergents (Japan); MTP (microtiter plate); MJ Research (MJ Research, Reno, Nev.); Baseclear (Baseclear BV, Inc., Leiden, The Netherlands); PerSeptive (PerSeptive Biosystems, Framingham, Mass.); ThermoFinnigan (ThermoFinnigan, San Jose, Calif.); Argo (Argo BioAnalytica, Morris Plains, N.J.); Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, N.Y. and Bad Kreuznach, Germany); Spectrum (Spectrum Laboratories, Dominguez Rancho, Calif.); Molecular Structure (Molecular Structure Corp., Woodlands, Tex.); Accelrys (Accelrys, Inc., San Diego, Calif.); Chemical Computing (Chemical Computing Corp., Montreal, Canada); New Brunswick (New Brunswick Scientific, Co., Edison, N.J.); CFT (Center for Test Materials, Vlaardingen, The Netherlands); P&G and Proctor & Gamble (Proctor & Gamble, Inc., Cincinnati, Ohio); GE Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom); DNA2.0 (DNA2.0, Menlo Park, Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (Megazyme International Ireland Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco, Wilmington, Del.); Corning (Corning Life Sciences, Corning, N.Y.); (NEN (NEN Life Science Products, Boston, Mass.); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, Tex.); ATCC (American Type Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Piscataway, N.J.); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, Calif.); BD Biosciences and/or Clontech (BD Biosciences CLONTECH Laboratories, Palo Alto, Calif.); Operon Technologies (Operon Technologies, Inc., Alameda, Calif.); MWG Biotech (MWG Biotech, High Point, N.C.); Oligos Etc (Oligos Etc. Inc, Wilsonville, Oreg.); Bachem (Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (Difco Laboratories, Detroit, Mich.); Mediatech (Mediatech, Herndon, Va.; Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); Oxoid (Oxoid Inc., Ogdensburg, N.Y.); Worthington (Worthington Biochemical Corp., Freehold, N.J.); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica, Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (Invitrogen Corp., San Diego, Calif.); NEB (New England Biolabs, Beverly, Mass.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pierce (Pierce Biotechnology, Rockford, Ill.); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); Gene Oracle (Gene Oracle, Inc., Mountain View, Calif.); EM Science (EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Biodesign (Biodesign Intl., Saco, Me.); Aptagen (Aptagen, Inc., Herndon, Va.); Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville, N.C.); Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.); R&D Systems (R&D Systems, Minneapolis, Minn.); Siegfried Handel (Siegfried Handel AG, Zofingen, Switzerland); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Marsh (Marsh Biosciences, Rochester, N.Y.); Geneart (Geneart GmbH, Regensburg, Germany); Bio-Tek (Bio-Tek Instruments, Winooski, Vt.); (Biacore (Biacore, Inc., Piscataway, N.J.); PeproTech (PeproTech, Rocky Hill, N.J.); SynPep (SynPep, Dublin, Calif.); New Objective (New Objective brand; Scientific Instrument Services, Inc., Ringoes, N.J.); Waters (Waters, Inc., Milford, Mass.); Matrix Science (Matrix Science, Boston, Mass.); Dionex (Dionex, Corp., Sunnyvale, Calif.); Monsanto (Monsanto Co., St. Louis, Mo.); Wintershall (Wintershall AG, Kassel, Germany); BASF (BASF Co., Florham Park, N.J.); Huntsman (Huntsman Petrochemical Corp., Salt Lake City, Utah); Shell Chemicals (Shell Chemicals, Inc., London, UK); Stepan (Stepan, Northfield, Ill.); Clariant (Clariant, Sulzbach, Germany); Industrial Zeolite (Industrial Zeolite Ltd., Grays, Essex, UK); Jungbunzlauer (Jungbunzlauer, Basel, Switzerland); Solvay (Solvay, Brussels, Belgium); 3V Sigma (3V Sigma, Bergamo, Italy); Innospec (Innospec, Ellesmere Port, UK); Thermphos (Thermphos, Vlissiggen-Ost, The Netherlands); Ciba Specialty (Ciba Specialty Chemicals, Basel, Switzerland); Dow Corning (Dow Corning, Barry, UK); Enichem (Enichem Iberica, Barcelona, Spain); Fluka Chemie AG (Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV, Delft, The Netherlands); Dow Corning (Dow Corning Corp., Midland, Mich.); Mettler-Toledo (Mettler-Toledo Inc, Columbus, Ohio); RB (Reckitt-Benckiser, Slough, UK); and Microsoft (Microsoft, Inc., Redmond, Wash.).

For North American (NA) and Western European (WE) heavy duty liquid laundry (HDL) detergents, heat inactivation of the enzymes present in commercially-available detergents is performed by placing pre-weighed liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The incubation time for heat inactivation of NA and WE auto dish washing (ADW) detergents is 8 hours. Both un-heated and heated detergents are assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity is tested by the AAPF assay.

For testing of enzyme activity in heat-inactivated detergents, working solutions of detergents are made from the heat inactivated stocks. Appropriate amounts of water hardness (e.g., 6 gpg or 12 gpg) and buffer are added to the detergent solutions to match the desired conditions. The solutions are mixed by vortexing or inverting the bottles. The following Table provides information regarding some of the commercially-available detergents and test conditions used herein. In some experiments, additional and/or other commercially available detergents find use in the following Examples.

TABLE 1.1 Laundry and Dish Washing Conditions Region Form Dose Detergent* Buffer gpg pH T (° C.) Laundry (Heavy Duty Liquid and Granular) NA HDL 0.78 g/l   P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0 g/L Henkel PERSIL ™ 5 mM HEPES 12 8.2 40 WE HDG 8.0 g/L P&G ARIEL ® 2 mM Na₂ CO₃ 12 10.5 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mM Na₂ CO₃ 6 10.0 20 NA HDG 1.0 g/L P&G TIDE ® 2 mM Na₂ CO₃ 6 10.0 20 Automatic Dish Washing WE ADW 3.0 g/L RB CALGONIT ™ 2 mM Na₂ CO₃ 21 10.0 40 NA ADW 3.0 g/L P&G CASCADE ® 2 mM Na₂ CO₃ 9 10.0 40

In some additional aspects, the following solutions find use:

TABLE 1-2 Working Detergent Solutions Temp Detergent Detergent (C.) g/L pH Buffer gpg TIDE ® 2X Cold 16 0.98 8 5 mM HEPES 6 TIDE ® 2X Cold 32 0.98 8 5 mM HEPES 6 TIDE ® 2X Cold 16 0.98 7 5 mM MOPS 6

The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The foregoing examples and detailed description are offered by way of illustration and not by way of limitation.

BPN′ Variant or Part I Examples Table of Detergents

The compositions of the detergents used in the assays in BPN′ Variant (or Part I) Examples are shown in Table 1-3. BPN′ variant protein samples were added to the detergent compositions as described in Part I Example 1 to assay for the various properties tested.

The following are liquid laundry detergent compositions suitable for top-loading automatic washing machines (1, 2 & 4) and front loading washing machines (3).

TABLE 1-3 Composition of Detergents Used in the Assays to Test BPN′ Variants Composition (wt % of composition) Ingredient 1 2 3 4 C₁₂₋₁₅ Alkylethoxy(1.8)sulfate 14.7 11.6 16.31 C_(11.8) Alkylbenzene sulfonate 4.3 11.6 8.3 7.73 C₁₆₋₁₇ Branched alkyl sulfate 1.7 1.29 3.09 C₁₂₋₁₄ Alkyl-9-ethoxylate 0.9 1.07 1.31 C₁₂ dimethylamine oxide 0.6 0.64 1.03 Citric acid 3.5 0.65 3 0.66 C₁₂₋₁₈ fatty acid 1.5 2.32 3.6 1.52 Sodium Borate (Borax) 2.5 2.46 1.2 2.53 Sodium C₁₂₋₁₄ alkyl ethoxy 3 sulfate 2.9 C₁₄₋₁₅ alkyl 7-ethoxylate 4.2 C₁₂₋₁₄ Alkyl-7-ethoxylate 1.7 Ca formate 0.09 0.09 0.09 A compound having the following general structure: 1.2 bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n), wherein n = from 20 to 30, and x = from 3 to 8, or sulphated or sulphonated variants thereof Random graft co-polymer¹ 1.46 0.5 Ethoxylated Polyethylenimine ² 1.5 1.29 1.44 Diethylene triamine pentaacetic acid 0.34 0.64 0.34 Diethylene triamine penta(methylene phosphonic acid) 0.3 Tinopal AMS-GX 0.06 Tinopal CBS-X 0.2 0.17 0.29 Amphiphilic alkoxylated grease cleaning polymer ³ 1.28 1 0.4 1.93 Ethanol 2 1.58 1.6 5.4 Propylene Glycol 3.9 3.59 1.3 4.3 Diethylene glycol 1.05 1.54 1.15 Polyethylene glycol 0.06 0.04 0.1 Monoethanolamine 3.05 2.41 0.4 1.26 NaOH 2.44 1.8 3.01 Sodium Cumene Sulphonate 1 Sodium Formate 0.11 0.09 Water, Aesthetics (Dyes, perfumes) and Minors balance balance balance (Enzymes, solvents, structurants) ¹Random graft copolymer is a polyvinyl acetate grafted polyethylene oxide copolymer having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is about 6000 and the weight ratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60 and no more than 1 grafting point per 50 ethylene oxide units. ² Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH. ³ Amphiphilic alkoxylated grease cleaning polymer is a polyethylenimine (MW = 600) with 24 ethoxylate groups per —NH and 16 propoxylate groups per —NH

Vectors, Cells, and Methods for Making Protease Variant Polypeptides of the Invention

A variety of methods are known in the art that are suitable for generating modified polynucleotides of the invention that encode protease variants of the invention (such as cold water proteases of the invention), including, but not limited to, e.g., site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches. Methods for making modified polynucleotides and proteins (e.g., protease variants) include DNA shuffling methodologies (see, e.g., Stemmer W P, Proc. Natl. Acad. Sci. USA 91(22):10747-51 (1994)); methods based on non-homologous recombination of genes, e.g., ITCHY (Ostermeier et al., Bioorg. Med. Chem. 7:2139-44 [1999]); SCRATCHY (Lutz et al. Proc. Natl. Acad. Sci. USA 98:11248-53 [2001]); SHIPREC (Sieber et al., Nat. Biotechnol. 19:456-60 [2001]); NRR (Bittker et al., Nat. Biotechnol. 20:1024-9 [2001]; Bittker et al., Proc Natl. Acad. Sci. USA101:7011-6 [2004]); methods that rely on the use of oligonucleotides to insert random and targeted mutations, deletions and/or insertions (Ness et al., Nat. Biotechnol. 20:1251-5 [2002]; Coco et al., Nat. Biotechnol. 20:1246-50 [2002]; Zha et al., Chembiochem. 4:34-9 [2003]; Glaser et al., J. Immunol. 149:3903-13 [1992]); see also Arkin and Youvan, Biotechnology 10:297-300 (1992); Reidhaar-Olson et al., Methods Enzymol. 208:564-86 (1991).

In one aspect, a full-length parent polynucleotide is ligated into an appropriate expression plasmid, and the following mutagenesis method is used to facilitate the construction of the modified protease of the present invention, although other methods may be used. The method is based on that described by Pisarchik et al. (Pisarchik et al., Prot. Eng. Des. Select. 20:257-265 [2007]). In one aspect, an added advantage is provided in that the restriction enzyme cuts outside its recognition sequence, which allows digestion of practically any nucleotide sequence and precludes formation of a restriction site scar.

In one approach, a naturally-occurring gene encoding a full-length protease is obtained and sequenced and scanned for one or more points at which it is desired to make a mutation (e.g., deletion, insertion, substitution) at one or more amino acids. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by primer extension in accord with generally known methods. Fragments to the left and to the right of the desired point(s) of mutation are amplified by PCR and to include the Eam1104I restriction site. The left and right fragments are digested with Eam1104I to generate a plurality of fragments having complementary three base overhangs, which are then pooled and ligated to generate a library of modified sequences containing one or more mutations. This method avoids the occurrence of frame-shift mutations. This method also simplifies the mutagenesis process because all of the oligonucleotides can be synthesized so as to have the same restriction site, and no synthetic linkers are necessary to create the restriction sites as is required by some other methods.

Part I Examples Example 1 Assays

Various assays were used as set forth below. Any deviations from the protocols provided below are indicated in the subsequent Examples.

A. TCA Assay for Protein Content Determination in 96-Well Microtiter Plates

For BPN′ and BPN′ variants, this assay was started using filtered B. subtilis bacterial culture supernatant from microtiter plates grown 3-4 days at 33-37° C. with shaking at 230-250 rpm and humidified aeration. A fresh 96-well flat bottom microtiter plate (MTP) was used for the assay. First, 100 μL/well of 0.25 N HCl was placed in each well. Then, 25 μL of filtered culture broth was added. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined in order to provide the “blank” reading. For the test, 100 μL/well of 30% (w/v) trichloroacetic acid (TCA) was placed in the plates and incubated for 10 minutes at room temperature. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined. The equipment used was a Biomek FX Robot (Beckman Coulter) and a SpectraMAX (type 340; Molecular Devices) MTP Reader; the MTPs were from Costar (type 9017).

The calculations were performed by subtracting the blank (no TCA) from the test reading with TCA to provide a relative measure of the protein content in the samples. If desired, a standard curve can be created by calibrating the TCA readings with AAPF assays of clones with known conversion factors. However, the TCA results are linear with respect to protein concentration from 250 to 2500 micrograms protein per ml (ppm) and can thus be plotted directly against enzyme performance for the purpose of choosing good-performing variants. The turbidity/light scatter increase in the samples correlates to the total amount of precipitable protein in the culture supernatant.

B. AAPF Protease Assay in 96-Well Microtiter Plates

In order to determine the protease activity of the proteases and variants thereof of the present invention, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mM Tris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer); 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl₂ and 0.005% TWEEN®-80 (Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1 ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer and mixed well for at least 10 seconds. The assay was performed by adding 10 μl of diluted protease solution to each well, immediately followed by the addition of 190 μl 1 mg/ml suc-AAPF-pNA working solution. The solutions were mixed for 5 sec., and the absorbance change in kinetic mode (25 readings in 5 minutes) was read at 405 nm in an MTP reader, at 25° C. The protease activity was expressed as AU (activity=ΔOD·min⁻¹ ml⁻¹).

C. BMI Microswatch Assay of Test Method 3

Blood milk and ink (BMI) stained microswatches of 5 5 millimeter circular diameter were obtained from CFT. Before cutting the swatches, the fabric (EMPA 116) was washed with water. One microswatch was placed in each well of a 96-well non-binding microtiter plate (Corning 3641). The detergents used for the assays included Detergent Composition 1, Detergent Composition 2, and Detergent Composition 4. The detergents were diluted in Milli-Q (deionized) water to a working strength concentration of 0.788 g/L. These detergents were buffered with 5 mM HEPES pH 8.2 or pH 7.2, which upon addition to detergent, buffers at pH 8 or pH 7, respectively. Additionally, 6 grains per gallon (gpg) water hardness (3:1 Ca:Mg—CaCl₂:MgCl₂.6H₂O) was added. The detergent solution was pre-equilibrated in an ice-water bath for 16° C. assays (room temperature for 32° C. assays) and pumped into a circulating reservoir (Beckman FX). Then, 190 μl of the desired detergent solution was added to each well of the MTP that contained microswatches. To this mixture, 10 μl of the diluted enzyme master dilution solution was added, providing an approximate enzyme concentration of 0.4-0.5 μg/mL. The master dilution was prepared from the culture supernatants at 8 μg/mL, where the approximate enzyme concentration of the culture supernatants and BPN′-v3 or BPN′-v36 parent controls was determined using the AAPF protease activity assay, basing the concentration on a purified BPN′-v3 or BPN′-v36 standard of known concentration. The MTP was sealed with tape and placed in the iEMS incubator/shaker (Thermo/Labsystems) pre-set at 16° C. in a refrigerated dairy case or at 32° C. on the benchtop for 20 minutes, with agitation at 1400 rpm. Following incubation under the appropriate conditions, the sealing tape was removed from each plate and 125 μl (150 μl if pipetting by hand for smaller screens) of the solution from each well was transferred into a fresh MTP (Corning 9017). The new MTP containing 125 μl-150 μl of solution/well was read at 600 nm (with 5 sec mixing mode in the plate reader) using a MTP SpectraMax reader (type 340; Molecular Devices). Blank controls containing a microswatch and detergent but no enzyme were also included. The absorbance value obtained was corrected for the blank value (substrate without enzyme), providing a measure of hydrolytic activity. For each sample (variant), the performance index was calculated as described below. This BMI Microswatch Assay, run at 60° F. (16° C.) and pH 8, is referred to herein as “Test Method. 3”

D. Stability Assay

The stability of protease variants was determined in the presence of 40% concentrated Detergent Composition 3 diluted in water. The reagents used were Detergent Composition 3 diluted to 50% in Milli-Q water, 10 mM MES 0.01% TWEEN®-80 pH 5.8 master dilution buffer, AAPF reagents: see protocol AAPF assay. The equipment used was F-bottom MTP (Corning 9017) for dilution of diluted enzyme into detergent as well as for suc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), sealing tape: Nunc (236366), circulating reservoir (Beckman Fx).

Detergent Composition 3 was initially diluted to 50% in water. This detergent was kept at room temperature and cycled through the circulating reservoir. The iEMS incubators/shakers (Thermo/Labsystems) were pre-set at 43° C. Culture supernatants were diluted into plates containing master dilution buffer to a concentration of ˜20 ppm (master dilution plate). Then, 40 μl of sample from the master dilution plate was added to plates containing 160 μl 50% Detergent Composition 3 to give a final incubation concentration of 4 ppm. The contents were mixed and kept at room temperature and triplicate AAPF assays were performed immediately on these plates and recorded as unstressed reads. The AAPF assay was modified such that 20 μL of sample from the step above was added to 190 μL of suc-AAPF-pNA working solution. The plates were immediately covered with sealing tape and placed in 43° C. iEMS shakers for 30 min at 650 rpm. Following 30 minutes of incubation, triplicate AAPF assays were performed on these stress plates and recorded as stressed reads. The stability of the samples was determined by calculating the ratio of the residual and initial AAPF activity as follows: Residual Activity (%)=[mOD·min⁻¹ stressed]*100/[mOD·min⁻¹ unstressed]. For each sample (variant), the performance index was calculated as described below.

E. LAS/EDTA Stability Assay

The stability of protease variants in the presence of a representative anionic surfactant (LAS=linear alkylbenzene sulfonate, specifically, sodium dodecylbenzenesulfonate-DOBS) and di-sodium EDTA was measured after incubation under defined conditions and the residual activity was determined using the AAPF assay. The reagents used were dodecyllbenzene sulfonate, sodium salt (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No. P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No. H-7523), unstressed buffer: 50 mM HEPES (11.9 g/l)+0.005% TWEEN®-80, pH 8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0, reference protease and protease variant culture supernatants, containing 200-400 μg/ml protein. The equipment used was V- or U-bottom MTPs as dilution plates (Greiner 651101 and 650161, respectively), F-bottom MTPs (Corning 9017) for unstressed and LAS/EDTA buffer as well as for suc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), and Nunc sealing tape (236366).

The iEMS incubator/shaker (Thermo/Labsystems) was set at 29° C. Culture supernatants were diluted into plates containing unstressed buffer to a concentration of ˜25 ppm (master dilution plate). Then, 20 μl of sample from the master dilution plate was added to plates containing 180 μl unstressed buffer to give a final incubation concentration of 2.5 ppm. The contents were mixed and kept at room temperature and an AAPF assay was performed on this plate. Then, 20 μl of sample from the master dilution plate was also added to plates containing 180 μl stress buffer (50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0). The solutions were mixed and immediately placed in 29° C. iEMS shaker for 30 min at 400 rpm. Following 30 minutes of incubation, an AAPF assay was performed on the stress plate. The stability of the samples was determined by calculating the ratio of the residual and initial AAPF activity as follows: Residual Activity (%)=[mOD·min−1 stressed]*100/[mOD·min−1 unstressed]. For each sample (variant), the performance index was calculated as described below.

Performance Index

The performance index provides a comparison of the performance of a variant (actual value) and a standard or reference protease enzyme (theoretical value) at the same protein concentration. The theoretical values can be calculated using the parameters of a performance dose response curve (i.e. using a Langmuir equation to generate the performance curve) of the standard/reference protease. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant as compared to the standard or reference protease (which may be, e.g., wild-type protease or another protease variant), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard or reference protease, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard or reference protease. Thus, the PI identifies winners (e.g., variants having enhanced proteolytic activity compared to that of the standard/reference protease) as well as variants that may be less desirable for use under certain circumstances (e.g., variants having proteolytic activity lower than the proteolytic activity of the standard/reference protease).

It is important to note that protease variants of the invention having performance index values lower than that of a reference or standard protease are nevertheless useful in the applications and methods described herein. For example, protease variants of the invention having performance index values lower than that of a reference or standard protease have proteolytic activity and thus are useful in the compositions of the invention, such as, but not limited to, e.g., cleaning compositions (including, but not limited, to, e.g., detergent cleaning compositions) for cleaning a variety of surfaces and items, including, but not limited to, e.g., laundry, fabrics, and dishware, and in personal care applications and compositions as described elsewhere herein; such protease variants are also useful in fabric and home care products and compositions and in non-fabric and home care products and compositions described elsewhere herein and in methods of the invention, including, but not limited, to, e.g., cleaning methods, methods for personal care, etc., described elsewhere herein.

Various terms set forth below are used to describe the variant: non-deleterious variants have a PI>0.05; deleterious variants have a PI less than or equal to 0.05; combinable variants are those for which the variant has performance index values greater than or equal to 0.2 for at least one property, and >0.05 for all properties. Combinable variants are those that can be combined to deliver proteins with appropriate performance indices for one or more desired properties. These data find use in engineering any subtilisin/subtilase or protease. Even if the subtilase or protease to be engineered has an amino acid different from that of subtilisin BPN′ at one or more particular positions, these data find use in identifying amino acid substitutions that alter the desired properties by identifying the best choices for substitutions, including substitutions of the BPN′ wild type amino acid.

Example 2 Construction of BPN′ Library and Cleaning Performance of BPN′ Variants

a) Description of the BPN′-v3 Expression Cassette Used for Library Construction

The BPN′-v3 (BPN′ protease containing G097A-G128A-Y217Q substitutions) expression cassette used for combinatorial library construction was generated using the BPN′ expression cassette, which comprises the aprE-BPN′ hybrid leader sequence (i.e., signal sequence), BPN′ pro and BPN′ mature sequence from B. amyloliquefaciens. The DNA sequence is shown below as SEQ ID NO:1 and encodes the BPN′ precursor protein shown below as SEQ ID NO:168.

(SEQ ID NO: 1) GTGAGAAGCAAAAAATTGTGGATCAGTTTGCTGTTTGCTTTAGCGTTAAT CTTTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAAT CAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACG ATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGT GCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAA AAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAA GATCACGTAGCACATGCGTAC GCGCAGTCCGTGCCTTACGGCGTATCACA AATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTA AAGTAGCGGTTATCGACAGCGGTATCGACTCGAGCCATCCAGATCTTAAA GTCGCTGGAGGGGCTTCTATGGTGCCGTCCGAAACAAACCCGTTTCAAGA TAACAATTCTCATGGCACACACGTCGCAGGAACGGTTGCGGCGTTAAACA ATTCTATTGGCGTGCTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTT AAAGTTCTTGGCGCAGACGGATCAGGCCAATACTCATGGATTATCAACGG CATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGAGCCTGG GAGGACCAAGCGGCAGTGCGGCACTTAAAGCAGCAGTTGATAAAGCTGTT GCATCTGGTGTCGTCGTAGTAGCGGCAGCTGGGAATGAGGGAACATCCGG ATCATCGAGTACCGTCGGTTATCCAGGCAAGTACCCTTCAGTGATTGCAG TGGGCGCTGTAGACTCTTCAAATCAACGTGCCTCTTTTTCCTCCGTGGGA CCGGAGCTGGATGTCATGGCCCCTGGCGTTTCTATTCAATCGACGCTTCC AGGGAACAAGTATGGTGCGTATAACGGGACTTCCATGGCCTCGCCGCATG TAGCTGGGGCGGCCGCATTGATTCTTTCTAAGCACCCGAACTGGACAAAC ACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTC TTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAG

In the nucleotide sequence of SEQ ID NO:1, the DNA sequence encoding the mature protease is shown in bold, the nucleotide sequence encoding leader sequence (aprE-BPN′ hybrid leader sequence) is shown in standard (non-underlined) text, and the nucleotide sequence encoding the pro sequence (BPN′) is underlined. In the amino acid sequence (aprE-BPN′ hybrid leader sequence, BPN′ pro sequence, and BPN′ mature protein sequence) of the BPN′ precursor protein set forth in SEQ ID NO:168, the bolded portion indicates the mature BPN′ subtilisin protease.

(SEQ ID NO: 168) VRSKKLWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIVGFKQTMST MSAAKKKDVISEKGGKVQKQFKYVDAASATLNEKAVKELKKDPSVAYVEE DHVAHAYAQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLK VAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAV KVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAV ASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVG PELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTN TQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ

The amino acid sequence of the mature BPN′ subtilisin protease is shown as SEQ ID NO:2.

The nucleotide sequence of the mature BPN′-v3 gene is that of SEQ ID NO:3 (the signal sequence and propeptide sequence used in the BPN′-v3 expression cassette is the same as that for BPN′ shown in SEQ ID NO:1). The protein sequence of the mature BPN′-v3 protease variant is that of SEQ ID NO:4 (the signal sequence and propeptide sequence used in the BPN′-v3 expression cassette is the same as that for BPN′ shown in SEQ ID NO:168).

b) Construction of Combinatorial Library Using pHPLT-BPN′-v3 Plasmid

The pHPLT-BPN′-v3 plasmid (see FIG. 1) containing the BPN′-v3 expression cassette described above served as template DNA for cloning to provide variants derived from BPN′-v3. The vector pHPLT (FIG. 4 in U.S. Pat. No. 6,566,112) contains the B. licheniformis LAT promoter (“Plat”); a sequence encoding the LAT signal peptide (“preLAT”). Additional plasmid elements from plasmid pUB110 disclosed in McKenzie et al., Plasmid 15(2): 93-103 (1986): “ori-pUB” is the origin of replication from pUB110; “neo” is the neomycin/kanamycin resistance gene from pUB110; “Terminator” is the transcriptional terminator from B. licheniformis amylase.

A combinatorial DNA library was synthesized at DNA 2.0 and delivered as individual ligation reactions. In some instances for efficient transformation of B. subtilis, the DNA from the ligation reaction mixtures was amplified by rolling circle amplification (RCA) using the Illustra Templiphi kit (GE Healthcare). The reaction was performed according to the manufacturer's protocol. One microliter of ten-fold diluted amplified DNA was used to transform 50 μL of competent B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). The transformation mixture was shaken at 37° C. for 1 hour. Ten micro-liter aliquots of the transformation mixture were plated on skim milk (1.6%) Luria agar plates supplemented with 10 μg/ml of neomycin (Teknova).

The transformants that formed halos on the skim milk plates were picked into microtiter plates containing 150 μl Luria broth (LB) medium supplemented with 10 μg/ml neomycin. Plates were grown overnight at 37° C. with 250-300 rpm shaking and 70-80% humidity using Enzyscreen lids for microtiter plates (Enzyscreen). Using a 96 pin replicating tool, (Enzyscreen) the overnight culture plate was used to inoculate a new microtiter plate containing 180 μl of MBD medium (a MOPS based defined medium) with 2.5 μg/ml neomycin. MBD medium was prepared essentially as known in the art (see Neidhardt et al., J. Bacteriol. 119:736-747 [1974]), except that NH₄Cl, FeSO₄, and CaCl₂ were omitted from the base medium, 3 mM K₂HPO₄ was used, and the base medium was supplemented with 60 mM urea, and 100 ml of a solution made of 210 g/L glucose, and 350 g/L maltodextrin. 1 g/L of BD Bacto Yeast Extract was added and the pH was adjusted to 7.4 with KOH. The micronutrients were made up as a 100× stock solution containing in one liter, 400 mg FeSO₄.7H₂O, 100 mg MnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100 mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂, and 10 ml of 0.5 M sodium citrate. The MBD medium containing microtiter plates were grown for 64 hours at 37° C., 250-300 rpm, and 70-80% humidity using Enzyscreen lids (Enzyscreen) for protease variant expression. The next day, cultures were filtered through a micro-filter plate (0.22 um; Millipore) and the resulting filtrates containing protease variants were used for biochemical analysis.

The protease variants were tested for cleaning performance using a BMI microswatch assay in Detergent Composition 1 at 16° C. and pH 8 and BMI microswatch assay in Detergent Composition 2 at 16° C. and pH 8. Protein content was determined using the TCA assay. Assays were performed as described in Example 1 and Performance Indices were calculated relative to BPN′-v3 (with a PI value of 1) as per Test Method 3.

The following BPN′ subtilisin protease variant was determined to have a PI value greater than 1.0, at least 1.1, or from greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising the set of amino acid substitutions G097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variant has a PI value of 1.1 relative to BPN′-v3 in this BMI microswatch cleaning assay, and enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) in this assay, the variant having an amino acid sequence comprising amino acid substitutions G097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Also included is a protease variant having enhanced proteolytic activity compared to SEQ ID NO:2 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising amino acid substitutions G097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value greater than that of BPN′ (SEQ ID NO:2) and/or BPN′-v3 in a BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises substitutions X097A-X128A-X210S-X217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2, and optionally wherein the variant comprises at least one substitution selected from the group of G097A, G128A, P210S, and Y217Q. Such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising such protease variant and methods for cleaning utilizing such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 (i.e., having a PI value approximately equivalent to that of BPN′-v3) in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128A-E156S-P210S-Y217Q, G097A-G128A-P210S-Y217Q-N218A, G097A-G128A-P210S-Y217Q-N218S, and G097A-Y104F-G128A-E156S-P210I-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value of 1.0 compared to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X097A, X104F, X128A, X156A/S, X210I/S, X217Q, X218A/S, and optionally at least one substitution selected from the group of G097A, Y104F, G128A, E156A/S, P210I/S, Y217Q, and N218A/S, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-E156A-P210S-Y217Q-N218S and G097A-G128A-Y217Q-N218A, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-P210S-Y217Q-N218A and G097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value of greater than 1 compared to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X097A, X104F, X128A, X156A/S, X210I/S, X217Q, X218A/S, and optionally at least one substitution selected from the group of G097A, Y104F, G128A, E156A/S, P210I/S, Y217Q, and N218A/S, wherein amino acid positions of the variant are numbered by correspondence with position of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q (i.e., BPN′-v3), G097A-G128A-E156S-P210S-Y217Q, and G097A-G128A-P210S-Y217Q-N218S, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity and enhanced proteolytic activity compared to BPN′ in this assay. The invention includes a protease variant having proteolytic activity, PI value of 1.0 relative to BPN′-v3, and/or enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-E156A-P210S-Y217Q-N218S, G097A-G128A-Y217Q-N218A, and G097A-Y104F-G128A-E156S-P210I-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 3 Generation of Combinatorial Libraries and Cleaning Performance of Variants of BPN′-v3+S78N

a) Description of BPN′-v3+S78N Variant and Synthetic Gene Sequences Derived from this Variant

Gene Oracle synthesized and cloned eight genes into the pHPLT-BPN′-v3+S78N (BPN′-S78N-G97A-G128A-Y217Q) parent plasmid (see FIG. 2). Some of these genes were used as templates (parents) to create combinatorial libraries. The BPN′-v3+S78N variant was generated using standard molecular biology methods known in the art. The nucleotide sequence encoding the BPN′-v3+S78N variant is that of SEQ ID NO:7 and the amino acid sequence of the BPN′-v3+S78N variant is that of SEQ ID NO:8. The nucleotide and protein sequences of genes GcM90-96, and GcM100 are shown below.

Genes Nucleotide Sequence Protein Sequence GcM90 SEQ ID NO: 9 SEQ ID NO: 10 GcM91 SEQ ID NO: 11 SEQ ID NO: 12 GcM92 SEQ ID NO: 13 SEQ ID NO: 14 GcM93 SEQ ID NO: 15 SEQ ID NO: 16 GcM94 SEQ ID NO: 17 SEQ ID NO: 18 GcM95 SEQ ID NO: 19 SEQ ID NO: 20 GcM96 SEQ ID NO: 21 SEQ ID NO: 22 GcM100 SEQ ID NO: 23 SEQ ID NO: 24

b) Construction of Combinatorial Libraries CG1-CG5 and CG8 Using the Synthetic Genes GcM90-94 and GcM100

TABLE 3-1 List of Possible Substitutions Introduced and Primers Used for the Construction of Combinatorial Libraries CG1-CG5 and CG8 Synthesized Genes (template or Library Substitutions Primer parent) Name Introduced Name Primer Sequence GcM90 CG1 G53S 1 S53 f /5Phos/CTTCTATGGTGCCGTCCGAAA CAAACCCGTTTCAAG (SEQ ID NO: 25) A68V 1 V68 f /5Phos/TCATGGCACACACGTCGCAGG AACGGTTGCGGCG (SEQ ID NO: 26) A102G 1 G102 f /5Phos/AGCAGACGGATCAGGCCAAT ACTCATGGATTATCAAC (SEQ ID NO: 27) T129P 1 P129 f /5Phos/TGAGCCTGGGAGCACCAAGC GGCAGTGCGGCACTTAAAG (SEQ ID NO: 28) T185Q 1 Q185 f /5Phos/TAGACTCTTCAAATCAACGTG CCTCTTTTTCCTCCGTG (SEQ ID NO: 29) GcM91 CG2 V59Q 2 Q59 f /5Phos/GAAACAAACCCGTTTCAAGAT AACAATTCTCATG (SEQ ID NO: 30) V108I 2 I108 f /5Phos/ATACTCATGGATTATCAACGG CATCGAATGGGCCATC (SEQ ID NO: 31) Q147V 2 V147 f /5Phos/TGTTGCATCTGGTGTCGTCGT AGTAGCGGCAGCTGG (SEQ ID NO: 32) A211G 2 G211 f /5Phos/ATCGACGCTTCCAGGGAACA AGTATGGTGCGCAAAAC (SEQ ID NO: 33) Q252N 2 N252 f /5Phos/CAGCAGTTTAGAAAACACCA CTACAAAACTTGGTG (SEQ ID NO: 34) GcM92 CG3 A61N 3 N61 f /5Phos/CAAACCCGTTTCAAGATAACA ATTCTCATGGCACACAC (SEQ ID NO: 35) E87S 3 S87 f /5Phos/TTGGTGTAGCCCCGTCTGCTT CGCTCTACGCCGTTAAAG (SEQ ID NO: 36) I124M 3 M124 f /5Phos/TGGATGTAATCAACATGAGCC TGGGAGCACCAAGCG (SEQ ID NO: 37) P161S 3 S161 f /5Phos/AGGGAACATCCGGATCATCG AGTACCGTCGGTTATCCAG (SEQ ID NO: 38) A224S 3 S224 f /5Phos/GACTTCCATGGCCTCGCCGCA TGTAGCTGGGGCGGC (SEQ ID NO: 39) GcM93 CG4 Q62N 4 N62 f /5Phos/GTTTCAAGATAACAATTCTCA TGGCACACACGTCGC (SEQ ID NO: 40) N100G 4 G100 f /5Phos/GTTCTTGCAGCAGACGGATCA GGCCAATACTCATG (SEQ ID NO: 41) A125S 4 S125 f /5Phos/ATGTAATCAACATGAGCCTGG GAGCACCAAGCGGCAG (SEQ ID NO: 42) D159S 4 S159 f /5Phos/GGAATGAGGGAACATCCGGA TCATCGAGTACCGTCGG (SEQ ID NO: 43) S240N 4 N240 f /5Phos/CTTTCTAAGCACCCGAACTGG ACAAACACTCAAGTCCG (SEQ ID NO: 44) GcM94 CG5 T63S 5 S63 f /5Phos/TCAAGATAACAATTCTCATGG CACACACGTCGCAGG (SEQ ID NO: 45) A101S 5 S101 f /5Phos/TGCAGCAGACGGATCAGGCC AATACTCATGGATTATC (SEQ ID NO: 46) V126L 5 L126 f /5Phos/AATCAACATGAGCCTGGGAG CACCAAGCGGCAGTG (SEQ ID NO: 47) T183S 5 S183 f /5Phos/CGCTGTAGACTCTTCAAATCA ACGTGCCTCTTTTTCC (SEQ ID NO: 48) N244T 5 T244 f /5Phos/GAACTGGACAAACACTCAAG TCCGCAGCAGTTTAG (SEQ ID NO: 49) GcM100 CG8 A68V 1 V68 f /5Phos/TCATGGCACACACGTCGCAGG AACGGTTGCGGCG (SEQ ID NO: 50) A102G 1 G102 f /5Phos/AGCAGACGGATCAGGCCAAT ACTCATGGATTATCAAC (SEQ ID NO: 51) A211G 2 G211 f /5Phos/ATCGACGCTTCCAGGGAACA AGTATGGTGCGCAAAAC (SEQ ID NO: 52) A125S 4 S125 f /5Phos/ATGTAATCAACATGAGCCTGG GAGCACCAAGCGGCAG (SEQ ID NO: 53)

Each synthesized gene was built into the pHPLT-BPN′-S78N-G97A-G128A-Y217Q parent molecule. Resulting plasmids containing the six synthesized genes GcM90-94, and GcM100 served as templates to make combinatorial libraries at the respective positions (Table 3-1). Two additional genes, GcM95 and GcM96, were also synthesized for analysis, but did not serve as parental DNA for libraries. These genes each have nine mutations on top of the pHPLT-BPN′-S78N-G97A-G128A-Y217Q parent molecule.

The parent plasmids (template DNA) containing the synthetic genes GcM90-94, and GcM100 were methylated were methylated using two micrograms of DNA and methylase (NEB), according to the NEB protocol. Methylated DNA was then purified using DNA Clean and Concentrator kit (Zymo Research). Combinatorial libraries CG1-5 and CG8 were made using a QUIKCHANGE® Multi Site-Directed Mutagenesis kit (“QCMS kit”; Stratagene) following the manufacturer's protocol (see Table 3-1 for respective template and primer combinations), with the exception of libraries CG3 and CG4, which used 86.5 ng of each primer in place of the 50 ng suggested in the protocol. All primers used for introducing the desired substitutions in each library are listed in Table 3-1. They were synthesized and provided by Integrated DNA Technologies. After the QCMS reactions were completed for each library, the template DNA was digested by the addition of 0.5-1 μl DpnI (from the QCMS kit) and incubated at 37° C. for 1-4 hours, followed by another addition of 0.5-1 μl DpnI and another incubation at 37° C. for 1-4 hours. For efficient transformation of B. subtilis, DNA from the QCMS reaction mixtures were amplified before transformation and transformants grown as described in Example 2.

Additional variants of BPN′-v3+S78N were produced by DNA2.0. The following substitutions were introduced individually into the BPN′-v3+S78N parent molecule: Q59G, N62Q, V68A, S89Y, A92G, I108V, I115V, M124T, P129L, A138T, V147L, S161P, Y167A, P172V, G211T, L267V, and A273S.

All of the combinatorial library variants described above and the variants synthesized at DNA2.0 were tested for cleaning performance using a BMI microswatch assay of Test Method 3 in Detergent Composition 1 at 16° C. and pH 8 and BMI microswatch assay in Detergent Composition 2 at 16° C. and pH 8. Protein content was determined using the TCA assay. Assays were performed as described in Example 1 and Performance Indices were calculated relative to the enzyme of SEQ ID NO:4 (with a PI value of 1.0).

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to the enzyme of SEQ ID NO:4 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S063T-S078N-G097A-S101A-G128A-S183T-Y217Q-T244N, N061A-S078N-G097A-G128A-Y217Q-S224A, S053G-S078N-G097A-G128A-P129T-Q185T-Y217Q, S063T-S078N-G097A-S101A-G128A-S183T-Y217Q, S063T-S078N-G097A-S101A-G128A-Y217Q, S063T-S078N-G097A-S101A-G128A-Y217Q-T244I, and S078N-G097A-G128A-P129T-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ and BPN′-v3 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X040E, X053G, X059V, X061A, X062H/Q, X068A, X078N, X087E, X101A, X102A, X108V, X124I, X125A, X126V, X129T, X147Q, X159D, X183T, X185T, X211A, X224A, X244I/N, X252Q, and X274D, and optionally at least one substitution selected from the group of X040E, X053G, X059V, X061A, X062H/Q, X068A, X078N, X087E, X101A, X102A, X108V, M124I, S125A, L126V, P129T, V147Q, S159D, S183T, Q185T, G211A, S224A, T244I/N, N252Q, and A274D, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to the enzyme of SEQ ID NO:4 (alternatively referred to as BPN′-v3) in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, N061A-S078N-S087E-G097A-G128A-Y217Q-S224A, Q059V-S078N-G097A-G128A-G211A-Y217Q, Q059V-S078N-G097A-G128A-V147Q-Y217Q, Q059V-S078N-G097A-G128A-Y217Q, Q059V-S078N-G097A-I108V-G128A-Y217Q-N252Q, S053G-S078N-G097A-G128A-P129T-Y217Q, S078N-G097A-G128A-G211A-Y217Q, S078N-G097A-G128A-Q185T-Y217Q, S078N-G097A-G128A-V147Q-Y217Q, S078N-G097A-G128A-Y217Q, S078N-G097A-G128A-Y217Q-S224A, and S078N-G097A-G128A-Y217Q-S224A-A274D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variant was determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising the set of amino acid substitutions S078N-G097A-I108V-G128A-V147Q-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising said set of amino acid substitutions above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of Q059V-S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q, S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q, S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q, S078N-G097A-I108V-G128A-V147Q-Y217Q-N252Q, and S078N-S087E-G097A-M124I-G128A-Y217Q-S224A, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S063T-S078N-G097A-S101A-G128A-S183T-Y217Q, S063T-S078N-G097A-S101A-G128A-S183T-Y217Q-T244N, S063T-S078N-G097A-S101A-G128A-Y217Q, and S063T-S078N-G097A-S101A-G128A-Y217Q-T244I, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, N061A-S078N-G097A-G128A-Y217Q-S224A, N061A-S078N-S087E-G097A-G128A-Y217Q-S224A, Q059V-S078N-G097A-G128A-G211A-Y217Q, Q059V-S078N-G097A-G128A-V147Q-Y217Q, Q059V-S078N-G097A-G128A-Y217Q, Q059V-S078N-G097A-I108V-G128A-Y217Q-N252Q, S053G-S078N-G097A-G128A-P129T-Q185T-Y217Q, S053G-S078N-G097A-G128A-P129T-Y217Q, S078N-G097A-G128A-G211A-Y217Q, S078N-G097A-G128A-P129T-Y217Q, S078N-G097A-G128A-Q185T-Y217Q, S078N-G097A-G128A-Y217Q, S078N-G097A-G128A-Y217Q-S224A, and S078N-G097A-G128A-Y217Q-S224A-A274D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of about 1 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S078N-G097A-G128A-V147Q-Y217Q and S078N-G097A-I108V-G128A-V147Q-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including cleaning compositions, comprising at least one such variant and methods for cleaning an item or surface in need of cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of Q059V-S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q, S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q, S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q, S078N-G097A-I108V-G128A-V147Q-Y217Q-N252Q, and S078N-S087E-G097A-M124I-G128A-Y217Q-S224A, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including cleaning compositions, comprising at least one such variant and methods for cleaning an item or surface in need of cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 4 Generation and Cleaning Performance of BPN′ Variants Generation of BPN′ Variants LC1-LC4 via QUIKCHANGE® Multi Site-Directed Mutagenesis

BPN′ variants were constructed from different parental plasmids using QUIKCHANGE® Multi Site-Directed Mutagenesis kits. The parental plasmids (Table 4-1) were methylated using a NEB Dam Methylase Kit in a reaction containing 77.5 μL H₂O+10 μL Buffer 10×+0.25 μL SAM+2 uL DAM methylase+10 uL miniprep DNA (˜150 ng/μL) at 37° C. overnight. The methylated plasmid DNA was purified using a QIAGEN® PCR purification kit. QUIKCHANGE® Multi Site-Directed Mutagenesis reactions were set up for each of the DNA templates in a reaction mix containing 2.5 μL Buffer 5×+0.5 μL primer 1 (25 μM)+0.5 μL primer 2 (25 μM)+1 μL dNTP's+1 μL enzyme blend+18 μL H₂O+1.5 μL DNA. The PCR program used was: 95° C. for 1 min; (95° C. for 1 min, 53° C. for 1 min, 65° C. for 9:39 min)×29 cycles; 65° C. for 10 min, 4° C. hold. Primer sequences are shown in Table 4-2. In all reactions, PCR was performed using a MJ Research PTC-200 Peltier thermal cycler. Parental DNA from the PCR samples was removed by addition of 1 μL of DpnI to QUIKCHANGE® Multi Site-Directed Mutagenesis reactions at 37° C. overnight. To increase transformation frequency, the DpnI-digested reactions were amplified using rolling circle amplification (RCA) using the Illustra TempliPhi kit according to the manufacturer's protocol. B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) were transformed with 1 μL each of the RCA reaction and the transformed cells were plated onto LA+1.6% skim milk plates containing 10 ppm neomycin and incubated at 37° C. overnight. Colonies from overnight growth were selected to perform colony PCR for sequencing using “puReTaq Ready-To-Go PCR Beads” (Amersham) The PCR and sequencing primers used were pHPLT F1 (/5PHOS/TACATATGAGTTATGCAGTTTG (SEQ ID NO:54)) and pHPLT seq R1 (/5PHOS/TTATCCTTTACCTTGTCTC (SEQ ID NO:55)). Clones with appropriate sequences were frozen. BPN′ variant proteins were produced by growing B. subtilis transformants in 96 well microtiter plates at 37° C. for 68 hours in a MOPS based medium containing urea as described in Example 2.

TABLE 4-1 Parental Plasmids and Primers Used for Generation of BPN′ Variants LC1-LC4 Mutations Parental Plasmid Introduced Primers Used BPN′-G097A-G128A-Y217Q-S024G- A128S A128Sf, A128Sr N025G-N061P-S101N (termed LC1) BPN′-G097A-G128A-Y217Q-S053G- A128S A128Sf, A128Sr N061P-S101N-V203Y (termed LC2) BPN′-G097A-G128A-Y217Q-S024G- A128S A128Sf, A128Sr N025G-S053G-T055P-N061P-S101N- V203Y (termed LC3) BPN′-G097A-G128A-Y217Q-S024G- P55T P55Tf, P55Tr N025G-S053G-T055P-N061P-S101N- V203Y (termed LC4)

TABLE 4-2 Sequences of Primers Used for QUIKCHANGE ® Multi Site-Directed Mutagenesis Reactions to Make BPN′ variants LC1-LC4 Primer Name Primer Sequence (5′ to 3′) A128Sf /5Phos/CAACATGAGCCTGGGATCACCAAGCGGCAGTGCGG (SEQ ID NO: 56) A128Sr /5Phos/CCGCACTGCCGCTTGGTGATCCCAGGCTCATGTTG (SEQ ID NO: 57) P55Tf /5Phos/CTATGGTGCCGGGCGAAACAAACCCGTTTCAAGATCCG (SEQ ID NO: 58) P55Tr /5Phos/CGGATCTTGAAACGGGTTTGTTTCGCCCGGCACCATAG (SEQ ID NO: 59)

Generation of Additional BPN′ Variants LC5-LC37

An additional 33 BPN′ variants termed successively LC5 through LC37 were produced by DNA 2.0 using the BPN′ nucleic acid as the parent gene contained in the expression plasmid pHPLT-BPN′ partial opt (see FIG. 3). LC5 through LC37 BPN′ variants are as follows, respectively: BPN′-P52L-V68A-G97A-I111V, BPN′-I111V-M124V-Y167A-Y217Q, BPN′-Y104N-G128A-Y217Q, BPN′-M124V-Y167A-Y217Q, BPN′-I111V-M124V-Y217Q, BPN′-P52L-V68A-G97A, BPN′-G97A-I111V-M124V, BPN′-V68A-A92G-G97A, BPN′-G97A-I111V-M124V-Y167A-Y217Q, BPN′-P52L-V68A-I111V-Y217Q, BPN′-P52L-V68A-I111V, BPN′-V68A-A92G-I111V, BPN′-P52L-V68A-G97A-I111V-Y217Q, BPN′-V68A-G97A-I111V, BPN′-G97A-I111V-Y217Q, BPN′-G97A-I111V-M124V-Y167A, BPN′-S89Y-I111V-M124V, BPN′-V68A-S89Y-I111V, BPN′-V68A-A92G-Y217Q, BPN′-I111V-Y167A-Y217Q, BPN′-G97A-I111V-Y167A-Y217Q, BPN′-G97A-I111V-M124V-Y217Q, BPN′-V68A-I111V-Y167A-Y217Q, BPN′-I111V-G128A-Y217Q, BPN′-G97A-M124V-Y217Q, BPN′-V68A-Y167A-Y217Q, BPN′-I111V-M124V-Y167A, BPN′-N62Q-G97A-I111V, BPN′-G97A-M124V-Y167A-Y217Q, BPN′-G97A-L126A-Y217Q, BPN′-V68A-I111V-Y217Q, BPN′-S89Y-M124V-Y217Q, and BPN′-L96T-G97A-Y217Q. Plasmid pHPLT-BPN′ partial opt was also created by DNA 2.0.

Transformants were picked into microtiter plates and grown as described in Example 2. The variants were assayed for cleaning performance using a BMI microswatch assay in Detergent Composition 2 at 16° C. and pH 8. Protein content was determined using the TCA assay. The assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN′-v3 (with a PI value of 1.0).

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-I111V-M124V-Y217Q, G097A-I111V-Y167A-Y217Q, S024G-N025G-N061P-G097A-S101N-G128S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128A-V203Y-Y217Q, S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128S-V203Y-Y217Q, and V068A-A092G-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value of greater than 1.0 compared to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X052L, X053G, X055P, X061P, X062Q, X068A, X089Y, X092G, X096T, X097A, X101N, X104N, X111V, X124V, X126A, X128A/S, X167A, X203Y, and X217Q, and optionally at least one substitution selected from the group of S024G, N025G, P052L, S053G, T055P, N061P, N062Q, V068A, S089Y, A092G, L096T, G097A, S101N, Y104N, I111V, M124V, L126A, G128A/S, Y167A, V203Y, Y217Q, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128S-Y217Q, G097A-I111V-Y217Q, I111V-G128A-Y217Q, I111V-M124V-Y167A, I111V-M124V-Y217Q, L096T-G097A-Y217Q, N062Q-G097A-I111V, S053G-N061P-G097A-S101N-G128S-V203Y-Y217Q, S089Y-M124V-Y217Q, and V068A-I111V-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a greater PI value than BPN′, and a PI value of 1.0 compared to BPN′-v3 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-I111V-M124V, G097A-L126A-Y217Q, G097A-M124V-Y217Q, I111V-Y167A-Y217Q, M124V-Y167A-Y217Q, P052L-V068A-G097A, S089Y-I111V-M124V, V068A-A092G-G097A, V068A-A092G-I111V, V068A-G097A-I111V, V068A-S089Y-I111V, and Y104N-G128A-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-M124V-Y167A-Y217Q, V068A-Y167A-Y217Q, G097A-I111V-M124V-Y167A, I111V-M124V-Y167A-Y217Q, V068A-I111V-Y167A-Y217Q, G097A-I111V-M124V-Y167A-Y217Q, P052L-V068A-I111V, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Such variants have proteolytic activity. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 5 Cleaning Performance of BPN′ Variants

Variants based on parent BPN′ were made by DNA 2.0. The variants were grown as described in Example 2 and tested for cleaning performance on BMI microswatch assay in Detergent Composition 1 at 16° C. and pH 8, BMI microswatch assay in Detergent Composition 4 at 16° C. and pH 8, and egg microswatch assay in Detergent Composition 4 at 16° C. and pH 8. The protein content was determined using the TCA assay. The assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN′-v3 (with a PI value of 1.0).

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-S101N-G128A-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q, S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128A-P210S-Y217Q, G097A-G128S-P201S-Y217Q, G097A-I111V-M124I-Y217Q, G097A-I111V-M124V-P201S-Y217Q, G097A-N123Q-P210S-Y217Q, G097A-N123Q-Y217Q, N061P-G097A-G128A-P210S-Y217Q, N061P-G097A-G128S-Y217Q, N061P-G097A-I111V-M124V-Y217Q, N061P-G097A-N123Q-Y217Q, N061P-G097A-S101N-I111V-M124V-Y217Q, N061P-G097A-S101N-N123Q-Y217Q, N061P-G102A-P129S-Y217Q, N061P-N062Q-G097A-G100N-S101N-Y217Q, N061P-N062Q-G097A-G100N-Y217Q, N061P-N062Q-G097A-G100Q-P210S-Y217Q, N061P-N062Q-G097A-I111V-Y217Q, N061P-N062Q-G097A-S101N-I111V-Y217Q, N061P-S078N-G097A-I111V-M124I-Y217Q, N061P-S078N-G102A-I111V-P129S-Y217Q, N062Q-G097A-I111V-P201S-Y217Q, N062Q-G097A-I111V-Y217Q, N062Q-S078N-G097A-I111V-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q, S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q, S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q, S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q, S053G-N061P-G097A-G128S-Y217Q, S053G-N061P-G097A-M124I-Y217Q, S053G-N061P-G097A-S101N-I111V-M124V-Y217Q, S053G-N061P-G102A-P129S-P210S-Y217Q, S053G-N061P-G102A-P129S-Y217Q, S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q, S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q, S053G-N061P-S101N-G102A-P129S-Y217Q, S053G-S078N-G097A-I111V-G128S-Y217Q, S078N-G097A-G128S-Y217Q, S078N-G097A-I111V-M124V-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-M124I-Y217Q, N061P-G097A-M124V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q, N061P-N062Q-G097A-G100Q-S101N-Y217Q, N061P-N062Q-G097A-G100Q-Y217Q, N061P-N062Q-G100N-G102A-Y217Q, N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q, S053G-N061P-G097A-S101N-M124I-Y217Q, S053G-N061P-G097A-S101N-N123Q-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value of greater than 1.0 compared to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 or 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X053G, X061P, X062Q, X078N, X097A, X100D/N/Q, X101N, X102A, X111V, X123A/Q/V, X124I/V, X128A/S, X129S, X210S, X217Q, and optionally at least one substitution selected from the group of S024G, N025G, S053G, N061P, N062Q, S078N, G097A, G100D/N/Q, S101N, G102A, I111V, N123A/Q/V, M124I/V, G128A/S, P129S, P210S, and Y217Q, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-N123A-Y217Q, G097A-N123V-Y217Q, N061P-G102A-G128S-Y217Q, N061P-S101N-G102A-G128S-Y217Q, Y217Q, S078N-G097A-I111V-N123Q-Y217Q, and G102A-N123Q-Y217Q, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-G128S-Y217Q, N061P-G097A-S101N-G128A-P201S-Y217Q, N061P-N062Q-G097A-S101N-I111V-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q, and S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128A-P210S-Y217Q, G097A-G128S-P201S-Y217Q, G097A-I111V-M124I-Y217Q, G097A-I111V-M124V-P210S-Y217Q, G097A-N123Q-P210S-Y217Q, G097A-N123Q-Y217Q, N061P-G097A-G128A-P210S-Y217Q, N061P-G097A-I111V-M124V-Y217Q, N061P-G097A-M124V-Y217Q, N061P-G097A-N123Q-Y217Q, N061P-G097A-S101N-I111V-M124V-Y217Q, N061P-G102A-P129S-Y217Q, N061P-N062Q-G097A-G100N-S101N-Y217Q, N061P-N062Q-G097A-G100Q-Y217Q, N061P-N062Q-G097A-I111V-Y217Q, N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q, N061P-S078N-G097A-I111V-M124I-Y217Q, N061P-S078N-G102A-I111V-P129S-Y217Q, N062Q-G097A-I111V-P210S-Y217Q, N062Q-G097A-I111V-Y217Q, N062Q-S078N-G097A-I111V-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q, S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q, S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q, S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q, S053G-N061P-G097A-G128S-Y217Q, S053G-N061P-G102A-P129S-P210S-Y217Q, S053G-N061P-G102A-P129S-Y217Q, S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q, S053G-N061P-S101N-G102A-P129S-Y217Q, S053G-S078N-G097A-I111V-G128S-Y217Q, S078N-G097A-G128S-Y217Q, and S078N-G097A-I111V-M124V-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-M124I-Y217Q, N061P-G097A-S101N-N123Q-Y217Q, N061P-N062Q-G097A-G100N-Y217Q, N061P-N062Q-G097A-G100Q-P210S-Y217Q, N061P-N062Q-G097A-G100Q-S101N-Y217Q, N061P-N062Q-G100N-G102A-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q, S053G-N061P-G097A-M124I-Y217Q, S053G-N061P-G097A-S101N-I111V-M124V-Y217Q, S053G-N061P-G097A-S101N-M124I-Y217Q, S053G-N061P-G097A-S101N-N123Q-Y217Q, and S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-N123A-Y217Q, G097A-N123V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q, N061P-S101N-G102A-G128S-Y217Q, Y217Q, N061P-G102A-G128S-Y217Q, S078N-G097A-I111V-N123Q-Y217Q, and G102A-N123Q-Y217Q, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value greater than 1.0 to about 5 relative to BPN′-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at 16° C. and pH 8: BPN′ amino acid sequence (SEQ ID NO:2) comprising the set of amino acid substitutions N061P-G097A-S101N-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising amino acid substitutions N061P-G097A-S101N-G128A-P201S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, N061P-G102A-P129S-Y217Q, N062Q-G097A-I111V-P201S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q, N061P-G097A-G128A-P201S-Y217Q, G097A-G128S-P201S-Y217Q, S024G-N025G-S053G-N061P-S078N-G097A-S101N-I111V-G128S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128S-Y217Q, N061P-G097A-G128S-Y217Q, G097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value equal to or greater than 0.5 and equal to or less than 0.9 relative to BPN′-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-M124I-Y217Q, S053G-N061P-G097A-S101N-N123Q-Y217Q, S053G-N061P-G102A-P129S-P201S-Y217Q, G097A-I111V-M124V-P201S-Y217Q, G097A-N123Q-P210S-Y217Q, S053G-N061P-S101N-G102A-P129S-Y217Q, S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q, N061P-N062Q-G097A-S101N-I111V-Y217Q, N061P-N062Q-G097A-I111V-Y217Q, N062Q-G097A-I111V-Y217Q, N061P-G097A-S101N-I111V-M124V-Y217Q, G097A-N123Q-Y217Q, N061P-G097A-I111V-M124V-Y217Q, S078N-G097A-I111V-M124V-Y217Q, S053G-S078N-G097A-I111V-G128S-Y217Q, S078N-G097A-G128S-Y217Q, S053G-N061P-G097A-G128S-Y217Q, N061P-N062Q-G097A-G100N-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-N123Q-Y217Q, N061P-G097A-S101N-N123Q-Y217Q, N061P-N062Q-G097A-G100Q-P210S-Y217Q, N061P-G097A-N123Q-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-M124I-Y217Q, S053G-N061P-G097A-S101N-M124I-Y217Q, S053G-N061P-G097A-M124I-Y217Q, N061P-S078N-G097A-I111V-M124I-Y217Q, N061P-G097A-M124V-Y217Q, S024G-N025G-S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q, S024G-N025G-S053G-N061P-S101N-G102A-P129S-Y217Q, N061P-S078N-G102A-I111V-P129S-Y217Q, S053G-N061P-G102A-P129S-Y217Q, S024G-N025G-S053G-N061P-N062Q-G097A-S101N-I111V-Y217Q, N062Q-S078N-G097A-I111V-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-I111V-M124V-Y217Q, S053G-N061P-G097A-S101N-I111V-M124V-Y217Q, G097A-I111V-M124I-Y217Q, Y217Q, N061P-N062Q-G100N-G102A-Y217Q, S053G-N061P-N062Q-G097A-G100N-S101N-Y217Q, N061P-N062Q-G097A-G100N-S101N-Y217Q, N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q, N061P-N062Q-G097A-G100Q-Y217Q, N061P-S101N-G102A-G128S-Y217Q, G097A-N123V-Y217Q, G097A-N123A-Y217Q, G102A-N123Q-Y217Q, N061P-N062Q-G097A-G100Q-S101N-Y217Q, S078N-G097A-I111V-N123Q-Y217Q, N061P-N062Q-G097A-G100D-Y217Q, and N061P-G102A-G128S-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value of equal to or greater than 0.5 and equal to or less than 0.9 relative to BPN′-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value of greater than 1.0 and to about 5 compared to BPN′-v3 in this egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X053G, X061P, X062Q, X078N, X097A, X100D/N/Q, X101N, X102A, X111V, X123A/Q/V, X124I/V, X128A/S, X129S, X210S, and X217Q, and optionally at least one substitution selected from the group of S024G, N025G, S053G, N061P, N062Q, S078N, G097A, G100D/N/Q, S101N, G102A, I111V, N123A/Q/V, M124I/V, G128A/S, P129S, P210S, and Y217Q, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 6

Construction and Cleaning Performance of BPN′ Variants

A BPN′ combinatorial library based on BPN′ parent was made by DNA2.0 and delivered as a ligation reaction. For efficient transformation of B. subtilis, DNA from the ligation reaction mixtures was amplified before transformation and transformants grown as described in Example 2. These variants were tested for cleaning performance using BMI microswatch assay of Test Method 3 in Detergent Composition 1 and Detergent Composition 4 at 16° C. and pH 8 as well as egg microswatch assay in Detergent Composition 4 at 16° C. and pH 8. Protein content was determined using the TCA assay and protease activity was assayed using the AAPF assay. The assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN′-v3 (with a PI value of 1.0).

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q, N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-N061P-S078N-G128A-Y217Q, N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, N025G-S078N-G097A-S101N-G128A-Y217Q, N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-G128A-Y217Q, S024G-N025G-T055P-G097A-G128A-Y217Q, S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-N061P-G097A-G128A-Y217Q, S024G-S053G-N061P-S078N-G097A-G128A-Y217Q, S024G-S053G-T055P-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S101N-G128A-Y217Q, S024G-T055P-N061P-G097A-G128A-Y217Q, S053G-G097A-S101N-G128A-Y217Q, S053G-N061P-G097A-S101N-G128A-Y217Q-S249N, S053G-N061P-S078N-G097A-G128A-Y217Q, S053G-S078N-G097A-S101N-G128A-Y217Q,

S053G-T055P-G097A-S101N-G128A-Y217Q, S053G-T055P-N061P-S101N-G128A-Y217Q, S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, T055P-G097A-S101N-G128A-Y217Q, and T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128S-Y217Q, G097A-G128A-Y217Q, N025G-S078N-G097A-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q, S024G-G097A-S101N-G128A-Y217Q, S024G-I035V-T055P-N061P-S078N-G097A-Y217Q, S024G-N025G-N061P-S078N-G097A-S101N-G128A, S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q, S024G-N025G-S053G-N061P-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q, S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-S101N-G128A-Y217Q, S024G-N025G-T055P-G097A-S101N-G128A-Y217Q, S024G-N025G-T055P-N061P-S078N-G097A-Y217Q, S024G-N061P-G097A-S101N-G128A-Y217Q, S024G-S038G-S053G-S078N-S101N-G128A-Y217Q, S024G-S053G-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-S078N-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S024G-T055P-G097A-G128A-Y217Q, S024G-T055P-N061P-G097A-S101N-G128A, S024G-T055P-N061P-S078N-S101N-G128A-Y217Q, S024G-T055P-S078N-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q, T055P-N061P-G097A-A116S-G128A, and T055P-N061P-S078N-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ and/or a PI value of greater than 1.0 compared to BPN′-v3 in this BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X035V, X038G, X053G, X055P, X061P, X078N, X097A, X101N, X116S, X128A/S, X130G, X216Q, X217Q, and X249N, and optionally at least one substitution selected from the group of S024G, N025G, I035V, S038G, S053G, T055P, N061P, S078N, G097A, S101N, A116S, G128A/S, S130G, Y216Q, Y217Q, and S249N, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-T055P-N061P-S078N-S101N-G128A-Y217Q, S053G-G097A-S101N-G128A-Y217Q, and T055P-N061P-S078N-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in a BMI microswatch cleaning assay of Test Method 3 in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128S-Y217Q, G097A-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q, N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-N061P-S078N-G128A-Y217Q, N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, N025G-S078N-G097A-G128A-Y217Q, N025G-S078N-G097A-S101N-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q, N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q, S024G-G097A-S101N-G128A-Y217Q, S024G-I035V-T055P-N061P-S078N-G097A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-S078N-G097A-S101N-G128A, S024G-N025G-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q, S024G-N025G-S053G-N061P-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q, S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-S101N-G128A-Y217Q, S024G-N025G-T055P-G097A-G128A-Y217Q, S024G-N025G-T055P-G097A-S101N-G128A-Y217Q, S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N061P-G097A-S101N-G128A-Y217Q, S024G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-S038G-S053G-S078N-S101N-G128A-Y217Q, S024G-S053G-N061P-G097A-G128A-Y217Q, S024G-S053G-N061P-S078N-G097A-G128A-Y217Q, S024G-S053G-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-S078N-S101N-G128A-Y217Q, S024G-S053G-T055P-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S024G-S053G-T055P-N061P-S101N-G128A-Y217Q, S024G-T055P-G097A-G128A-Y217Q, S024G-T055P-N061P-G097A-G128A-Y217Q, S024G-T055P-N061P-G097A-S101N-G128A, S024G-T055P-S078N-G097A-S101N-G128A-Y217Q, S053G-N061P-G097A-S101N-G128A-Y217Q-S249N, S053G-N061P-S078N-G097A-G128A-Y217Q, S053G-S078N-G097A-S101N-G128A-Y217Q, S053G-T055P-G097A-S101N-G128A-Y217Q, S053G-T055P-N061P-S101N-G128A-Y217Q, S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q, T055P-G097A-S101N-G128A-Y217Q, and T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay of Tets Method 3 in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of T055P-N061P-G097A-A116S-G128A, S024G-N025G-T055P-N061P-S078N-G097A-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity and may have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) in this assay. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-G128A-Y217Q, S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-N061P-S078N-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-G097A-S101N-G128A-Y217Q, S053G-N061P-S078N-G097A-G128A-Y217Q, S024G-N025G-T055P-G097A-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.

The following BPN′ variants were determined to have a PI value equal to about 1.0 relative to BPN′-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128S-Y217Q, G097A-G128A-Y217Q, S024G-G097A-S101N-G128A-Y217Q, N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, S053G-T055P-N061P-S101N-G128A-Y217Q, S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-S078N-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, T055P-N061P-S078N-G128A-Y217Q, N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-G128A-Y217Q, S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q, T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-Y217Q, S024G-T055P-G097A-G128A-Y217Q, T055P-N061P-G097A-A116S-G128A, S053G-T055P-G097A-S101N-G128A-Y217Q, T055P-G097A-S101N-G128A-Y217Q, S024G-N061P-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q, S024G-S053G-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S053G-G097A-S101N-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q, S024G-T055P-S078N-G097A-S101N-G128A-Y217Q, N025G-S078N-G097A-S101N-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q, S024G-S053G-N061P-S078N-G097A-G128A-Y217Q, S024G-N025G-T055P-N061P-S078N-G097A-Y217Q, S024G-I035V-T055P-N061P-S078N-G097A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning comprising utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S101N-G128A-Y217Q, S024G-T055P-N061P-G097A-S101N-G128A, S024G-N025G-N061P-S078N-G097A-S101N-G128A, S024G-T055P-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S053G-N061P-G097A-S101N-G128A-Y217Q-S249N, N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-T055P-G097A-S101N-G128A-Y217Q, S024G-S053G-N061P-G097A-G128A-Y217Q, S024G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S024G-T055P-N061P-G097A-G128A-Y217Q, S024G-S038G-S053G-S078N-S101N-G128A-Y217Q, S053G-S078N-G097A-S101N-G128A-Y217Q, N025G-S078N-G097A-G128A-Y217Q, and S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variant was determined to have a PI value of about 0.8 relative to BPN′-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising amino acid substitutions S024G-S053G-S078N-G097A-S101N-G128A-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. The invention includes a protease variant having proteolytic activity, a PI value of 0.8 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising amino acid substitutions S024G-S053G-S078N-G097A-S101N-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1 to about 12, from greater than 4 to about 12, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v3 in an AAPF proteolytic assay: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S024G-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q, N025G-T055P-N061P-S078N-S101N-G128A-Y217Q, S053G-T055P-N061P-S101N-G128A-Y217Q, S024G-T055P-N061P-G097A-S101N-G128A, S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-S078N-G097A-S101N-G128A, N061P-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, S024G-T055P-N061P-S078N-S101N-G128A-Y217Q, N025G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-S101N-G128A-Y217Q, N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-G128A-Y217Q, S024G-S053G-T055P-N061P-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, T055P-N061P-S078N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-S038G-S053G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-G128A-Y217Q, N025G-S053G-N061P-S078N-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-S078N-S101N-G128A-Y217Q, T055P-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G128A-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-Y217Q, S053G-N061P-G097A-S101N-G128A-Y217Q-S249N, N025G-S053G-T055P-S078N-G097A-S101N-G128A-Y217Q, S024G-T055P-G097A-G128A-Y217Q, T055P-N061P-G097A-A116S-G128A, S024G-N025G-T055P-G097A-S101N-G128A-Y217Q, S024G-N025G-N061P-S078N-G097A-S101N-G128A-Y217Q, S053G-T055P-G097A-S101N-G128A-Y217Q, T055P-G097A-S101N-G128A-Y217Q, S024G-N061P-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-G097A-S101N-G128A-Y217Q, G097A-G128S-Y217Q, S024G-S053G-N061P-G097A-G128A-Y217Q, S024G-N025G-N061P-G097A-G128A-Y217Q, S024G-N061P-S078N-G097A-S101N-G128A-Y217Q, S024G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S024G-S053G-S078N-S101N-G128A-Y217Q, S024G-N025G-S053G-T055P-N061P-S078N-G097A-G128A-Y217Q, S053G-N061P-S078N-G097A-G128A-Y217Q, S024G-T055P-N061P-G097A-G128A-Y217Q, S024G-S038G-S053G-S078N-S101N-G128A-Y217Q, S053G-G097A-S101N-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q, S024G-T055P-S078N-G097A-S101N-G128A-Y217Q, S053G-S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-T055P-G097A-G128A-Y217Q, S024G-N025G-S053G-T055P-G097A-G128A-Y217Q, N025G-S078N-G097A-S101N-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q, S024G-S053G-N061P-S078N-G097A-G128A-Y217Q, S024G-S053G-S078N-G097A-S101N-G128A-Y217Q, N025G-S078N-G097A-G128A-Y217Q, and S024G-N025G-S053G-N061P-G097A-G128A-S130G-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ protease (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v3 in this AAPF assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.

The following BPN′ variant was determined to have a PI value equal to about 1.0 relative to BPN′-v3 in an AAPF proteolytic assay: BPN′ amino acid sequence (SEQ ID NO:2) comprising amino acid substitutions G097A-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ protease (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN′-v3 in this AAPF assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising amino acid substitutions G097A-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 7 Construction of Site Evaluation Libraries of BPN′-v36 and Cleaning Performance of BPN′-v36 Variants Construction of the Site Evaluation Libraries of BPN′-v36

The amino acid sequence of BPN′-v36 is that of SEQ ID NO:6 and the nucleic acid sequence encoding the BPN′-v36 protease variant is that of SEQ ID NO:5.

The amino acid sequence of BPN′-v36 may be represented by reference to the subtilisin BPN′ amino acid sequence of SEQ ID NO:2. That is, BPN′-v36 may be represented as the subtilisin BPN′ sequence of SEQ ID NO:2 with the six amino acid substitutions S024G-S053G-S078N-S101N-G128A-Y217Q. The BPN′-v36 amino acid sequence may be conveniently designated as BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q or BPN′+S024G+S053G+S078N+S101N+G128A+Y217Q. Throughout this specification, unless otherwise indicated, each amino acid position of an amino acid sequence is numbered according to the numbering of a corresponding amino acid position in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN′ shown in SEQ ID NO:2 as determined by alignment of the variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN′ amino acid sequence.

Site evaluation libraries (SELs) were created at every single amino acid position in mature BPN′-v36 (i.e., BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q) protein by PCR fusion.

For each codon to be mutated in the BPN′-v36 protease, a pair of partially overlapping, complementary (mutagenic forward and reverse) primers were designed. Each mutagenic primer contained the NNS (N=A,C,G, or T and S=G or C) mutagenic codon in the center flanked by at least 15 nucleotides on each side. To create a library at a given position, two PCR reactions were carried out using either a common forward gene-flanking primer (P4974, SEQ ID NO:60) and a mutagenic NNS reverse primer, or the common reverse gene-flanking primer (P4976, SEQ ID NO:61) and a mutagenic NNS forward primer. These PCR reactions generated two PCR fragments, one encoding the 5′ half of the mutant BPN′-v36 gene (5′ gene fragment) and the other encoding the 3′ half of the mutant BPN′-v36 gene (3′ gene fragment).

Each PCR amplification reaction contained 30 pmol of each primer and 100 ng of the BPN′-v36 parent template DNA (plasmid pHPLT-BPN′-v36, see FIG. 4) Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 40 sec. Following amplification, the 5′ and 3′ gene fragments were gel-purified by the QIAGEN® gel-band purification kit, mixed (50 ng of each fragment), mixed and amplified by PCR once again using the primers P4973 (SEQ ID NO:62) and P4950 (SEQ ID NO:63) to generate the full-length gene fragment. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min. The full-length DNA fragment was gel-purified by the QIAGEN® gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ partial opt vector that also was digested with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification in an Illustra Templiphi kit according to the manufacturer's recommendation (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, the colonies with halos were inoculated in 150 μl of LB media containing 10 μg/mL neomycin. The next day, cultures were either frozen with 15% glycerol or grown in MBD medium for biochemical analysis as described in Example 2.

Cleaning Performance of the BPN′-v36 Variants

Protein variants from BPN′-v36 SEL were tested for cleaning performance using a BMI microswatch assay in Detergent Composition 4 at 16° C. and pH 8 and egg microswatch assay in Detergent Composition 4 at 16° C. and pH 8. Protein content was determined using the TCA assay. The assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN′-v36 (i.e., BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q).

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q (SEQ ID NO:6) (i.e., BPN′-v36) comprising at least one amino acid substitution selected from the group consisting of A116V, G160S, I111L, I115V, N109S, N117M, P005G, Q059V, T164S, Y262M, A015Q, A015S, A098E, A098N, A098S, A098T, A098V, A098Y, A114S, A114T, A116G, A116L, A116S, A116T, A116W, A133G, A133H, A133T, A133V, A137G, A137I, A137L, A137S, A137T, A138S, A216E, A216F, A216V, D099S, D181E, F261A, F261Q, G024F, G024I, G024Q, G024Y, G097S, G160T, G211L, G211V, H017F, H017W, H039V, H226A, I031V, I111V, I268V, K170R, K265R, L016Q, L016T, L135M, L209T, L209V, L233M, L257T, L257V, L267A, L267V, N025A, N025I, N025Q, N025R, N025T, N025V, N101I, N101Q, N101S, N109A, N109G, N109H, N109L, N109M, N109Q, N109T, N117Q, N184A, N184L, N184T, N184W, N212G, N212L, N212V, N243P, N252G, N252M, P005T, P014S, P040G, P040L, P040Q, P129A, P129S, P172G, P172S, P194Q, P210A, P210S, Q185F, Q185G, Q185I, Q185M, Q185N, Q185S, Q275H, R186K, S009A, S009G, S009H, S009M, S018T, S130T, S132N, S145K, S159T, S161I, S161K, S161N, S161T, S162I, S162M, S162Y, S163G, S182F, S182G, S182V, S182W, S183F, S183L, S183M, S183T, S183V, S183W, S224A, S236T, S249V, T022A, T022G, T022Q, T022V, T208V, T242S, T253N, T253S, T254A, T254S, T255L, T255S, T255V, V004A, V004P, V004W, V084C, V139C, V165M, V203F, Y021K, Y021N, Y021T, Y021V, Y167F, Y171F, Y214F, Y262F, and Y262T, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′-v3 and BPN′-v36, a PI value greater than that of BPN′-v3, and/or a PI value greater than 1 to about 5 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of BPN′-v36, A001G, A001Y, A013G, A013V, A015F, A015G, A015K, A015M, A015P, A015T, A015W, A015Y, A029G, A073S, A088C, A088I, A088L, A088T, A088V, A098D, A098K, A098P, A098R, A098W, A116D, A116E, A116R, A128S, A133L, A133M, A133S, A134G, A134S, A137N, A137V, A144M, A144Q, A144S, A144T, A144V, A151C, A176S, A176T, A179S, A216G, A216L, A216P, A216Q, A216S, A216T, A216Y, A228T, A230C, A231C, A272I, A272L, A272Q, A272S, A272T, A272W, A273S, A274G, A274L, A274Q, A274T, A274V, D041E, D099G, D099N, D120A, D120K, D120Q, D120R, D120S, D140E, D181S, D259E, E054D, E156D, E156T, E251V, F261G, F261H, F261L, F261R, F261S, F261T, F261V, F261W, G007A, G007S, G020A, G020D, G020S, G024N, G024R, G024S, G024T, G024V, G024W, G053H, G053K, G053N, G053T, G097A, G097D, G097T, G131A, G131H, G131P, G131Q, G131T, G131V, G160H, G160P, G166A, G166S, G166T, G211A, G211D, G211M, G211N, G211P, G211Q, G211R, G211W, G215A, G215N, G215V, H017L, H017M, H017T, H017V, H017Y, H039A, H039C, H039N, H226F, H226I, H226M, H226S, H226V, H226Y, I035V, I079A, I079S, I079T, I079V, I079W, I108V, I115L, I122A, I234L, I234V, K012R, K027R, K136R, K141F, K213W, K237R, K256R, K265Q, L016A, L016F, L016I, L016S, L016V, L042I, L075A, L075M, L075Q, L075V, L075Y, L082K, L082M, L082Q, L082V, L090I, L196I, L196V, L209Q, L209W, L233A, L233Q, L233V, L235I, L235K, L250I, L257A, L257H, L257Q, L257S, L257Y, L267Q, L267R, L267S, L267T, M199V, N025F, N025G, N025H, N025K, N025L, N025M, N025S, N025Y, N061F, N061H, N061P, N061S, N061T, N061V, N061W, N076G, N076W, N078S, N078T, N078V, N101A, N101H, N101L, N101T, N109K, N109R, N117A, N117E, N117H, N117K, N118G, N184G, N184H, N184I, N184S, N184V, N212A, N212F, N212I, N212K, N212P, N212Q, N212S, N212Y, N218A, N218H, N218L, N218S, N240E, N240H, N240L, N240R, N240T, N243A, N243Q, N243T, N243V, N252A, N252K, N252L, N252Q, N252R, N252S, N252T, N269Q, N269S, P014G, P014Q, P014T, P040A, P040H, P040S, P040T, P040V, P040Y, P086A, P086C, P086F, P086H, P086S, P129D, P129G, P129K, P129T, P172A, P172Q, P194A, P194G, P194L, P194M, P194S, P194V, P194Y, P210G, P210R, P210V, Q002A, Q002S, Q010A, Q010F, Q010H, Q010I, Q010L, Q010N, Q010S, Q010T, Q019A, Q019G, Q019N, Q019S, Q019T, Q019V, Q019W, Q059I, Q103L, Q103S, Q185A, Q185H, Q185L, Q185T, Q185Y, Q206P, Q206S, Q206Y, Q217I, Q217N, Q217S, Q217T, Q245K, Q275D, Q275S, Q275W, S003A, S003G, S003H, S003M, S003P, S003Q, S003T, S003V, S009I, S009L, S009P, S009T, S009W, S018A, S018G, S018I, S018L, S018M, S018N, S018P, S018V, S018W, S033T, S037Q, S037T, S037V, S038G, S038H, S038K, S038Q, S038T, S063K, S063N, S063Q, S063T, S087A, S087F, S087G, S087Q, S087T, S089L, S089M, S089N, S089Q, S089T, S089W, S130A, S130F, S130G, S130L, S130V, S145A, S145H, S145M, S145V, S159A, S159G, S159H, S159Q, S159R, S161A, S161G, S161H, S161L, S161M, S161P, S161Q, S161W, S162A, S162F, S162G, S162L, S162N, S162P, S162R, S162V, S163P, S173A, S173G, S182A, S182H, S182K, S182L, S182N, S182P, S182Q, S182T, S183A, S183G, S183H, S183Q, S188A, S188G, S188T, S188V, S191A, S204A, S204I, S204L, S204Q, S204V, S224C, S236A, S236N, S236Q, S248A, S248F, S248G, S248I, S248K, S248L, S248M, S248N, S248Q, S248T, S248V, S249A, S249C, S249H, S249Q, S249T, S249W, S249Y, S260H, S260N, S260P, S260T, T022H, T022K, T022N, T022R, T022S, T022Y, T055A, T055G, T055L, T055N, T055P, T055Q, T071S, T158H, T158S, T164N, T208C, T208L, T220S, T242N, T244A, T244G, T244H, T244I, T244Q, T244S, T244V, T244W, T253A, T253G, T253H, T253Q, T254V, T255A, T255G, T255H, T255I, T255Q, T255Y, V004G, V004N, V004R, V008A, V008C, V008M, V026I, V044I, V044L, V045H, V045K, V045L, V045M, V045Q, V045S, V045V, V045W, V045Y, V051I, V081L, V081Q, V081T, V084A, V084S, V084T, V093I, V121I, V143N, V143S, V143Y, V147C, V147I, V147L, V147T, V180I, V180L, V180T, V192A, V192S, V192T, V198I, V198L, V198M, V203H, V203I, V203L, V203N, V203Q, V203T, V203W, V203Y, V270A, V270S, V270T, W241M, W241Y, Y006G, Y006H, Y006I, Y006K, Y006L, Y006P, Y006Q, Y006T, Y006V, Y006W, Y021A, Y021D, Y021E, Y021L, Y021Q, Y021R, Y021S, Y104F, Y104I, Y214L, Y214V, Y214W, Y262A, Y262G, Y262L, Y262N, Y262S, Y262W, Y263G, and Y263W, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Thus, e.g., the invention includes BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising substitution A128S, e.g., BPN′-S024G-S053G-S078N-S101N-G128S-Y217Q. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of about 1.0 relative to BPN′-v3, and/or a PI value of 1.0 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined using Test Method 3 to have a PI value equal to about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of A001F, A001K, A001L, A001M, A001Q, A001R, A001S, A001T, A001V, A013C, A013S, A015D, A015E, A015L, A015R, A048S, A073N, A073T, A074G, A074S, A085C, A085G, A085S, A085V, A088M, A088S, A092S, A098G, A114G, A133P, A137E, A137H, A144G, A144H, A144K, A144L, A144N, A153S, A153V, A176C, A179G, A187G, A187S, A200G, A216W, A223S, A228S, A230T, A230V, A231V, A232C, A232V, A272E, A272G, A272K, A272P, A273G, A273L, A273V, A274M, A274R, D036E, D099A, D099Q, D120E, D181A, D181G, D259A, D259G, D259Q, D259T, E156A, E156S, E251I, E251L, E251Q, E251T, F058Y, F261C, F261D, F261K, F261P, G020E, G020F, G020H, G020L, G020N, G020Q, G020R, G020T, G020Y, G024A, G024P, G053A, G053D, G053E, G053F, G053L, G053Q, G053S, G053Y, G097K, G097M, G157A, G157S, G160A, G160L, G166C, G166I, G166Q, G169A, G211K, G215H, G215L, G215S, G215T, G215W, G258S, H017I, H039S, H226L, H238N, H238Y, I011L, I011V, I031L, I079F, I079K, I079L, I079M, I079Q, I205A, I205V, I268L, I268M, K012G, K043F, K043H, K043I, K043N, K043Q, K043T, K141A, K141R, K141W, K170A, K213A, K213G, K213H, K213I, K213L, K213N, K213Q, K213R, K213S, K213T, K213V, K237A, K237H, K237I, K237L, K237N, K237S, K256A, K256G, K256H, K256M, K256P, K256Q, K256W, K265H, L016E, L042V, L075G, L075H, L075I, L075T, L082A, L082F, L082H, L082R, L082S, L082T, L090M, L135F, L196M, L209C, L209H, L209S, L233S, L235M, L235R, L235W, L257C, L257G, L267F, M050Y, M119C, M119I, M124L, N025C, N025E, N025P, N061A, N061G, N061I, N061K, N061L, N061Q, N061R, N062S, N062T, N076A, N076P, N076Q, N076S, N076T, N076V, N078G, N078H, N078K, N078P, N078Q, N078R, N101F, N117R, N117S, N118D, N118H, N118Q, N118R, N118S, N118T, N184C, N184E, N184R, N212D, N212R, N212W, N218F, N218G, N218M, N218P, N218T, N218V, N218W, N240A, N240G, N240Q, N240S, N240W, N243C, N243G, N243S, N252V, N269H, P005A, P005D, P005M, P005Q, P014A, P014M, P014R, P014V, P040F, P040R, P040W, P129E, P129R, P172E, P172K, P194H, P194R, P194W, P201A, P201G, P210L, P239K, P239R, Q002D, Q002E, Q002G, Q002I, Q002P, Q002V, Q010D, Q010R, Q019C, Q019D, Q019E, Q019H, Q019L, Q019P, Q019R, Q059A, Q059E, Q059L, Q059S, Q059T, Q103W, Q185D, Q185K, Q185R, Q185W, Q206G, Q206H, Q206L, Q206V, Q206W, Q217E, Q217F, Q217H, Q217L, Q217V, Q245M, Q271A, Q271D, Q271G, Q271L, Q271P, Q271T, Q271Y, Q275F, Q275L, Q275P, Q275R, S003D, S003F, S003K, S003R, S009K, S018D, S018R, S037A, S037G, S037K, S037L, S037P, S038M, S063A, S063F, S063G, S063M, S063R, S063Y, S087C, S087K, S087L, S087M, S087N, S087Y, S089A, S089D, S089F, S089G, S089H, S089I, S089K, S089R, S089V, S089Y, S130D, S130E, S130K, S130W, S145G, S145L, S145R, S145T, S159D, S159L, S159W, S161E, S161R, S162C, S162E, S162W, S163A, S182E, S182R, S183C, S183D, S183P, S183R, S188D, S188P, S204G, S204Y, S207G, S224G, S224T, S236C, S236G, S248D, S248H, S248R, S249E, S249L, S249R, S260A, S260G, S260K, S260Q, S260V, S260Y, T022L, T055D, T055E, T055I, T055K, T055M, T055S, T055V, T055Y, T158A, T158G, T158L, T158Q, T158V, T164K, T164Q, T208S, T244D, T244E, T244R, T253E, T253R, T253Y, T254G, T255D, T255E, T255K, T255R, V026A, V028I, V028L, V030I, V044C, V044P, V045E, V045G, V045N, V072L, V081A, V081G, V081H, V081S, V084I, V084M, V095A, V095C, V143A, V143F, V143H, V143Q, V143T, V143W, V147A, V147Q, V147S, V148I, V148L, V149C, V149I, V149L, V165L, V180A, V180C, V180M, V192C, V192F, V192I, V192Q, V192Y, V203A, V203G, V203K, V203S, V270C, V270L, V270P, W241F, Y006A, Y006M, Y006N, Y006R, Y006S, Y021C, Y091W, Y104V, Y104W, Y262C, Y262D, Y262E, Y262H, Y262I, Y262R, and Y262V, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants may have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and/or a greater PI value than BPN′ in this assay. The invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in the BMI microswatch cleaning assay of Test Method 3 in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of A001D, A001H, A001N, A015C, A048C, A048E, A085T, A133R, A137R, A142C, A144D, A144R, A152S, A153G, A187P, A187Q, A187T, A187V, A216R, A230S, A272R, A273H, A273T, A274H, D036N, D036S, D181H, D181T, D259N, D259P, D259S, E156G, E156H, E156L, E156Q, E156V, E251C, F189S, F189T, F189W, F189Y, F261E, G020C, G024D, G053M, G053R, G097R, G131D, G131R, G157N, G160R, G160V, G166L, G166W, G211E, G215D, G258A, G258D, G258P, I011T, I031C, I079E, I079R, I175L, I205C, K012H, K012N, K027A, K027N, K027S, K043A, K043D, K043E, K043G, K043L, K043M, K043P, K043V, K043W, K043Y, K136H, K141H, K141L, K141M, K141N, K141Q, K141T, K141V, K170G, K170S, K237T, K237V, K256D, K256S, K256T, K256V, K265N, K265S, L042F, L042M, L082E, L209A, L209E, L209G, L209R, L233G, L235V, L257D, L257E, L257P, L257R, L257W, L267E, M050L, N056D, N056S, N061C, N061D, N062A, N062H, N062L, N062V, N062Y, N076D, N076L, N076M, N078D, N078F, N101D, N101R, N118A, N212C, N212E, N218C, N218D, N218E, N252D, N252E, P014F, P014K, P057A, P057W, P172R, P194E, P201T, P210E, Q059C, Q059D, Q059R, Q185E, Q206D, Q217A, Q217K, Q217R, Q245A, Q245D, Q245E, Q245H, Q245R, Q271E, Q271F, Q271R, Q271W, Q275G, Q275I, R186I, R186L, R186V, R186W, S003E, S009C, S009E, S018C, S037D, S037E, S037H, S037R, S037Y, S038D, S038P, S038R, S038Y, S063L, S087D, S087R, S089C, S089E, S130C, S130R, S145D, S159C, S159P, S161C, S173T, S182C, S188E, S188F, S188K, S188L, S188R, S188W, S190A, S190G, S190T, S204R, S236D, S236E, S248C, S248E, S260C, S260E, S260R, T022P, T055C, T055W, T071A, T158D, T158E, T158P, T158R, T158Y, T164R, T242D, T242G, T255C, V004E, V004T, V045C, V045D, V045R, V045T, V051H, V081R, V143C, V143E, V143G, V192G, V203C, V203D, V203E, V203M, V203R, V270G, W241L, Y214H, Y214Q, A001E, A133E, A187L, A187N, A216C, A216H, A273Q, D099H, D259H, E156C, E195G, F189H, G131C, G146A, G166V, G215C, G215E, I107L, K012A, K012S, K012T, K043C, K170C, K256C, K256E, K265G, K265Y, L233E, M222F, M222S, N062Q, N076E, N078E, N184P, N218R, P005V, P014D, Q002K, Q002L, Q002R, Q010W, Q271C, R186H, S049C, S063C, S063D, S105T, S188C, S190C, S204E, T055R, T164G, V004D, V044T, V045I, V165C, V180S, Y006C, Y006D, Y006E, Y104T, A001C, A187C, A230G, A273D, A273P, D036Q, F189G, F189L, F189R, G157T, G178A, I031F, I111M, K012F, K012L, K027T, K043R, K136G, K141G, K170Q, M222A, M222L, N062R, N117G, N269C, P005W, P129V, P239A, P239H, P239T, Q059W, Q217G, Q275A, R186A, S191G, T164A, T220A, A001P, A187F, A187W, A273R, D041C, D060G, D197T, F189A, G046D, G157P, K012C, K012E, K012W, L042C, M222T, N062C, P239G, P239N, Q217C, R186M, S049T, S089P, S125A, S173V, and V044A, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this BMI microswatch cleaning assay(Test Method 3), the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at 16° C. and pH 8: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of A216E, L090I, A098R, A098W, A098Y, A116G, A116R, A116S, A133M, I107L, I115V, M124L, N101I, N109H, N109S, N109T, N117R, P005G, Q185L, S089V, V095A, A015Y, A029G, A098D, A098E, A098G, A098N, A098S, A098T, A098V, A114S, A114T, A116E, A116L, A116T, A116V, A133H, A133L, A133S, A137G, A137I, A137L, A137S, A137V, A138S, A144S, A144V, A176S, A176T, A187T, A216F, A216P, A216Q, A216R, A216S, A216T, A216V, A216Y, D041E, D120A, D120E, D120Q, D120R, D120S, D181S, G020A, G020S, G024A, G097A, G097D, G097S, G131Q, G160S, G166I, G211L, G215N, H039N, H238N, I111L, I111V, I122A, L075I, L075Q, L135M, L209T, L209V, L233V, L235M, L235R, L257A, M119I, N025A, N025G, N025T, N061K, N101F, N101H, N101L, N101Q, N101R, N101S, N101T, N109A, N109G, N109K, N109L, N117E, N117H, N117K, N117S, N212G, N212S, N218F, N218G, N218H, N218L, N218S, N218W, N240Q, N252M, N252R, N252S, P005T, P040A, P040G, P040T, P129D, P129S, P194S, P210R, Q019R, Q019W, Q103L, Q103W, Q185A, Q185G, Q185M, Q185R, Q185T, Q206G, Q206Y, Q217A, Q217E, Q217R, Q217S, Q217T, S003Q, S009H, S018M, S033T, S130A, S130F, S130G, S130T, S130V, S145T, S159A, S161N, S161T, S162V, S162Y, S182L, S182W, S183F, S183L, S183V, S183W, S188K, S188W, S236Q, S236T, S248L, T022H, T022K, T208C, T253H, T255V, V044I, V121I, V139C, V143H, V143Q, V143T, V143W, V143Y, Y006K, Y021A, Y104W, A001F, A001G, A001H, A001K, A001L, A001Q, A001S, A001Y, A013V, A015G, A015K, A015R, A015S, A015T, A015W, A048S, A073N, A073S, A092S, A098K, A098P, A116D, A116W, A128S, A133P, A133T, A133V, A134G, A134S, A137H, A137N, A137T, A144D, A144K, A144L, A144M, A144N, A144R, A179G, A179S, A187V, A216G, A216L, A216W, A223S, A230C, A272K, A272L, A272P, A272S, A272T, A272W, A273G, A273S, A274G, A274M, A274T, D120K, D140E, D181A, D181E, D181G, D181H, D181T, D259E, D259N, D259Q, E054D, E156D, E156T, E251L, E251T, E251V, F058Y, F189W, F261K, F261Q, F261R, G007A, G007S, G020F, G020H, G020N, G020Q, G020T, G020Y, G024F, G024Q, G024R, G024T, G024V, G024W, G024Y, G053T, G097K, G097M, G097R, G097T, G131A, G131H, G131P, G131R, G131T, G131V, G160H, G160T, G166C, G166Q, G166S, G166T, G211A, G211D, G211K, G211M, G211N, G211Q, G211R, G211V, G211W, G215S, G215T, G215W, H017T, H017W, H017Y, H039V, H226A, H226F, H226I, H226L, H226M, H226V, I035V, I079A, I079S, I108V, I205V, I234L, I234V, I268V, K012S, K043P, K136H, K136R, K141A, K141F, K141T, K141W, K170A, K170G, K170R, K213A, K213R, K213S, K237A, K237H, K237L, K237S, K237V, K256A, K256G, K256H, K256M, K256P, K256Q, K256R, L016A, L016Q, L016T, L016V, L042V, L075M, L075T, L082M, L082V, L135F, L196I, L209H, L209Q, L209R, L209S, L209W, L233A, L233M, L233Q, L235I, L235K, L250I, L257S, L257T, L257V, L267A, L267Q, L267T, L267V, M119C, M199V, N025C, N025E, N025F, N025I, N025K, N025L, N025M, N025Q, N025V, N025Y, N061F, N061P, N061S, N061T, N076G, N078S, N101A, N109M, N109Q, N109R, N117A, N117M, N117Q, N118D, N118G, N118H, N118Q, N118R, N118S, N184A, N184C, N184G, N184L, N184R, N184S, N184T, N184V, N184W, N212C, N212F, N212I, N212K, N212L, N212P, N212Q, N212R, N212V, N212W, N212Y, N218A, N218P, N218T, N240A, N240E, N240G, N240H, N240L, N240R, N240S, N240T, N243C, N243Q, N243T, N243V, N252A, N252G, N252K, N252Q, P014G, P014Q, P014R, P014S, P014T, P040F, P040L, P040Q, P040S, P040V, P086C, P086H, P086S, P129A, P129E, P129G, P129K, P129R, P172A, P172K, P172Q, P172S, P194A, P194G, P194H, P194L, P194M, P194Q, P194R, P194V, P194W, P194Y, P210A, P210G, P210L, P210S, P239K, P239R, Q002A, Q002S, Q010A, Q010N, Q010R, Q010T, Q019A, Q019C, Q019D, Q019G, Q019S, Q019T, Q019V, Q059I, Q059V, Q103S, Q185F, Q185H, Q185I, Q185K, Q185N, Q185S, Q185Y, Q206H, Q206L, Q206P, Q206W, Q217F, Q217H, Q217I, Q217K, Q217L, Q217N, Q217V, Q271G, Q271R, Q271T, Q275F, Q275P, Q275R, R186A, R186I, R186K, S003A, S003F, S003G, S003H, S003K, S003R, S003T, S009T, S018N, S018T, S037G, S037T, S037V, S038G, S038Q, S063N, S063Q, S063T, S089M, S089N, S130K, S130L, S130R, S130W, S132N, S145G, S145K, S145M, S145R, S145V, S159C, S159H, S159L, S159Q, S159R, S159T, S159W, S161A, S161C, S161G, S161H, S161I, S161K, S161P, S161Q, S161R, S162F, S162G, S162I, S162L, S162M, S162N, S162P, S162R, S163G, S173A, S173G, S182F, S182G, S182K, S182N, S182Q, S182V, S183A, S183M, S183Q, S183R, S183T, S188A, S188F, S188G, S188P, S188R, S188T, S188V, S190C, S204A, S204G, S204I, S204L, S204Q, S204R, S204V, S207G, S224A, S224T, S236C, S236D, S236E, S236G, S236N, S248A, S248F, S248K, S248M, S248T, S249A, S249R, S249T, S249V, S249W, S249Y, S260G, S260H, S260K, S260N, T022A, T022G, T022Q, T022S, T022V, T022Y, T055A, T055K, T158A, T158S, T208L, T208S, T208V, T220S, T242D, T242N, T242S, T244E, T244G, T244I, T244R, T244V, T244W, T253A, T253G, T253N, T253S, T254S, T254V, T255H, T255I, T255K, T255L, T255Q, T255R, T255S, T255Y, V004A, V004N, V004P, V004W, V008A, V008M, V026I, V045S, V045W, V051I, V081Q, V081T, V084C, V093I, V095C, V143A, V143E, V143F, V143N, V143S, V147I, V147L, V147S, V147T, V148I, V149C, V149I, V165M, V180A, V180C, V180I, V180L, V180T, V192A, V192C, V192I, V192S, V192T, V192Y, V198L, V203A, V203F, V203K, V203L, V203M, V203N, V203Y, W241Y, Y006A, Y006G, Y006H, Y006L, Y006N, Y006P, Y006Q, Y006T, Y021E, Y021K, Y021L, Y021N, Y021Q, Y021R, Y021S, Y021T, Y104F, Y104I, Y104V, Y171F, Y214F, Y214L, Y214W, Y262F, and Y262S, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′-v3 and BPN′-v36, a PI value greater than that of BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of BPN′-v36, A001D, A001M, A001N, A001R, A001T, A001V, A013C, A013G, A013S, A015D, A015F, A015L, A015M, A015P, A015Q, A074G, A074S, A085S, A085T, A085V, A088C, A088L, A088S, A088T, A088V, A133E, A133G, A133R, A137E, A144G, A144H, A144Q, A144T, A151C, A152S, A153G, A153S, A153V, A176C, A187G, A187Q, A187S, A200G, A228S, A228T, A230T, A230V, A231C, A231V, A232C, A232V, A272E, A272G, A272I, A272Q, A272R, A273L, A273V, A274L, A274Q, A274R, A274V, D036E, D259A, D259G, D259P, D259S, D259T, E156A, E156S, E156V, E195G, E251C, E251I, E251Q, F189Y, F261A, F261G, F261H, F261L, F261P, F261S, F261T, F261V, F261W, G020C, G020D, G020E, G020L, G020R, G024D, G024I, G024N, G024P, G024S, G053A, G053D, G053F, G053H, G053K, G053N, G053S, G053Y, G157A, G157S, G160A, G160L, G160P, G160R, G166A, G166L, G166V, G166W, G169A, G211E, G211P, G215A, G215D, G215E, G215H, G215V, G258D, G258S, H017F, H017I, H017L, H017M, H017V, H039C, H226S, H226Y, I011T, I011V, I031C, I031L, I031V, I079F, I079K, I079L, I079M, I079Q, I079R, I079T, I079V, I079W, I115L, I205A, I205C, I268L, K012R, K012T, K027R, K043A, K043E, K043F, K043H, K043I, K043M, K043N, K043Q, K043T, K043V, K043Y, K141H, K141Q, K141V, K170S, K213G, K213H, K213I, K213L, K213N, K213Q, K213T, K213W, K237I, K237N, K237R, K237T, K256C, K256E, K256S, K256T, K256V, K256W, K265H, K265R, L016E, L016F, L016I, L016S, L042I, L042M, L075A, L075H, L075Y, L082K, L082Q, L082T, L196M, L196V, L209A, L209C, L209E, L209G, L235W, L257C, L257H, L257Q, L257Y, L267E, L267F, L267R, L267S, M050Y, N025H, N025P, N025S, N056D, N061A, N061C, N061D, N061G, N061H, N061I, N061L, N061Q, N061R, N061V, N061W, N062A, N062S, N062V, N076A, N076E, N076L, N076M, N076Q, N076S, N076T, N076W, N078G, N078H, N078K, N078P, N078Q, N078T, N101D, N118A, N118T, N184E, N184H, N184I, N212A, N212D, N212E, N218C, N218D, N218E, N218M, N218R, N218V, N240W, N243A, N243G, N243P, N243S, N252D, N252E, N252L, N252T, N252V, N269S, P005A, P005D, P005M, P005Q, P014A, P014D, P014F, P014M, P014V, P040H, P040R, P040W, P040Y, P086A, P129T, P172E, P172G, P172R, P194E, P201A, P201G, P210E, P210V, Q002E, Q002G, Q010D, Q010F, Q010H, Q010I, Q010L, Q010S, Q019E, Q019H, Q019N, Q059A, Q059L, Q059R, Q059S, Q059T, Q185D, Q185E, Q206D, Q206S, Q206V, Q217G, Q245D, Q245E, Q245K, Q245M, Q245R, Q271A, Q271F, Q271P, Q271Y, Q275D, Q275H, Q275I, Q275L, Q275S, Q275W, R186H, R186L, R186V, R186W, S003D, S003E, S003M, S003P, S003V, S009A, S009E, S009G, S009I, S009K, S009M, S009P, S009W, S018A, S018G, S018I, S018L, S018P, S018R, S018V, S018W, S037A, S037D, S037Q, S037R, S037Y, S038D, S038H, S038K, S038M, S038R, S038T, S063A, S063G, S063K, S063M, S063R, S087A, S087D, S087F, S087G, S087Q, S087T, S089A, S089C, S089H, S089I, S089K, S089L, S089Q, S089R, S089T, S089Y, S130D, S130E, S145A, S145H, S145L, S159G, S161E, S161L, S161M, S161W, S162A, S162C, S162E, S162W, S163P, S173T, S182A, S182C, S182E, S182H, S182R, S182T, S183C, S183D, S183G, S183H, S188C, S188D, S188E, S188L, S190T, S191A, S204Y, S224C, S236A, S248C, S248D, S248G, S248I, S248N, S248Q, S248R, S248V, S249C, S249H, S249L, S249Q, S260A, S260C, S260E, S260P, S260Q, S260R, S260T, T022L, T022N, T022R, T055C, T055D, T055G, T055I, T055L, T055N, T055Q, T055S, T055V, T055Y, T071A, T071S, T158G, T158H, T158L, T158P, T158Q, T158R, T158V, T158Y, T164N, T164S, T244A, T244D, T244H, T244Q, T244S, T253Q, T253R, T253Y, T254A, T254G, T255A, T255D, T255E, V004E, V004G, V004R, V008C, V026A, V028L, V030I, V044C, V045D, V045E, V045H, V045M, V045Q, V045Y, V072L, V081L, V081S, V084A, V084I, V084M, V084T, V143C, V147C, V147Q, V149L, V165L, V180M, V192F, V192G, V192Q, V198I, V198M, V203C, V203E, V203H, V203I, V203Q, V203R, V203T, V203W, V270A, V270L, V270T, W241F, W241M, Y006C, Y006D, Y006E, Y006I, Y006M, Y006R, Y006S, Y006V, Y006W, Y021C, Y021D, Y021V, Y091W, Y167F, Y214V, Y262A, Y262C, Y262I, Y262M, Y262R, Y262T, Y262V, and Y262W, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of about 1.0 relative to BPN′-v3, and/or a PI value of 1.0 relative to BPN′-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein. The following BPN′-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of A001E, A015C, A015E, A048C, A048E, A073T, A085C, A085G, A088I, A088M, A114G, A137R, A187L, A187N, A187P, A187W, A216C, A230S, A273D, A273H, A273T, D036N, D099N, D259H, E156C, E156G, E156H, E156Q, F189H, F189R, F189S, F189T, F261C, F261D, F261E, G053E, G053M, G053Q, G131C, G131D, G157N, G160V, G215C, G215L, G258A, H039A, I011L, I079E, I268M, K012A, K012G, K012H, K012N, K027N, K043C, K043D, K043G, K043L, K043W, K136G, K141G, K141L, K141M, K141N, K141R, K170C, K170Q, K213V, K256D, K265G, K265N, K265Q, K265S, L082A, L082F, L082H, L082R, L082S, L090M, L233S, L235V, L257E, L257G, L257R, L257W, M222S, N025R, N062H, N062T, N076D, N076P, N078D, N078E, N078F, N078R, N078V, N269H, N269Q, P014K, P057A, P086F, P201T, Q002D, Q002I, Q002P, Q002V, Q019L, Q019P, Q059C, Q059D, Q059E, Q185W, Q271C, Q271D, Q271E, Q271L, Q271W, Q275G, R186M, S009C, S009L, S018D, S037E, S037H, S037K, S037L, S037P, S038P, S063C, S063D, S063F, S063L, S063Y, S087L, S087N, S087R, S087Y, S089D, S089F, S089G, S089W, S105T, S125A, S130C, S159D, S159P, S163A, S182P, S183P, S190A, S190G, S204E, S224G, S248E, S248H, S249E, S260V, S260Y, T055M, T055R, T055W, T158D, T158E, T164G, T164K, T164Q, T220A, T242G, T253E, T255C, T255G, V004D, V044L, V044P, V045C, V045G, V045L, V045N, V045R, V045V, V081A, V081G, V081H, V084S, V147A, V203D, V203G, V270C, V270P, V270S, W241L, Y104T, Y214Q, Y262D, Y262E, Y262G, Y262H, Y262L, Y262N, Y263G, and Y263W, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), and/or a PI value of 0.9 relative to BPN′-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of A001C, A142C, A187C, A216H, A273Q, A274H, D036Q, D036S, D099S, D197T, E156L, F189A, F189L, G053L, G053R, G157P, G178A, G258P, H039S, H238Y, K012C, K012E, K012L, K012W, K136E, K265Y, L075G, L075V, L082E, L126W, L257D, L257P, M050L, M222A, M222F, M222L, N056S, N062C, N062L, N062Y, N269C, P057W, Q002K, Q002L, Q217C, Q245A, Q245H, S018C, S038Y, S049C, S087C, S087K, S145D, S191G, T022P, T055E, T164A, T164R, V045K, V051H, V081R, V143G, V148L, V180S, V203S, V270G, Y214H, A187F, A273P, F189G, G046D, G146A, G157T, I031F, I175L, K012F, K027T, L042F, L233E, L233G, M222T, N062R, N184P, P005V, P005W, P129V, P239N, P239T, Q010W, Q059W, Q275A, V004T, V165C, A128H, A230G, D041C, H067T, K027S, K043R, L090T, N062Q, N117G, P225G, P225S, P239G, P239H, Q002R, S089E, V044A, V045I, A001P, A273R, D041N, D099A, D099H, D099Q, F058G, I111M, L042C, N118L, P239A, S049N, S089P, S173V, T242P, V044T, and V045T, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value of equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 8 Cleaning Performance of Additional Combinatorial Variants Based on BPN′-v36 Parent

Additional combinatorial variants based on parent BPN′-v36 (BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q) were made and provided by DNA 2.0. These variants were tested for their cleaning performance using BMI microswatch assay in Detergent Composition 4 at 16° C. and pH 8, BMI microswatch assay in Detergent Composition 4 at 16° C. and pH 7, Egg microswatch assay in Detergent Composition 4 at 16° C. and pH 8, and Grass microswatch assay in Detergent Composition 4 at 16° C. and pH 8. Protein content was determined using TCA assay and protease activity was assayed using AAPF assay. All assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN′-v36.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-L257G, A116T-A128S, N061S-N109G-A128S-N243V-S260P, S009T-N109G-A128S-K141R-N243V, S009T-S018T-Y021N-N109G-A128S-K141R, and S162G-K256R, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A088T, A088T-A116T, A088T-G131H, A088T-K256R, A088T-N109G, A088T-N243V, A088T-Q103H, A088T-S162G, A088T-S248N, A088T-S249A, A088T-T158S, A116T, A116T-G131H, A116T-K256R, A116T-L257G, A116T-N243V, A116T-S162G, A116T-S248N, A116T-S249A, A116T-T158S, A128S-K256R, A128S-L257G, A128S-N243V-S248N-K256R, A128S-S162G, A128S-S248N, A128S-S249A, A128S-T158S, G024E-A116T, G024E-K256R, G024E-L257G, G024E-N109G, G024E-N243V, G024E-T158S, G131H, G131H-K256R, G131H-L257G, G131H-N243V-K256R, G131H-S162G, G131H-S248N, G131H-S249A, G131H-T158S, K043Y-A088T, K043Y-K256R, K043Y-N243V, K256R, K256R-L257G, L257G, N061G-N109G-N243V, N061P-N109G-G131H-N243V, N061P-N109G-N243V, N061S-A128S-N243V-S260P, N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-A128S-S260P, N061S-N109G-N243V, N076D-K256R, N076D-L257G, N076D-N109G, N076D-T158S, N109A-A128S-N243V-K256R, N109G, N109G-A116T, N109G-A128S, N109G-A128S-G131H-N243V-S248N-K256R, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A, N109G-A128S-N243V-S248A-K256R, N109G-A128S-N243V-S248N, N109G-A128S-N243V-S248N-K256R, N109G-A128S-N243V-S248N-K256R-L257G, N109G-A128S-S162G-N243V-S248N-K256R, N109G-A128S-S248N-K256R, N109G-A128S-T158S-N243V-S248N-K256R, N109G-G131H, N109G-K256R, N109G-L257G, N109G-N218S, N109G-N243P-S248A-K256R, N109G-N243P-S248N-K256R, N109G-N243V, N109G-N243V-K256R, N109G-N243V-S248A-K256R, N109G-N243V-S248N, N109G-N243V-S248N-K256R, N109G-S162G, N109G-S248N-K256R, N109G-S249A, N109G-T158S, N109Q-A128S-N243V-K256R, N109S-A128S-N243V-K256R, N218S-N243V, N243V, N243V-K256R, N243V-L257G, N243V-S248N, N243V-S248N-K256R, N243V-S249A, P040A-N109G-A128S-N243V-S248N-K256R, Q103H-A116T, Q103H-A128S, Q103H-G131H, Q103H-K256R, Q103H-L257G, Q103H-N109G, Q103H-N218S, Q103H-N243V, Q103H-S162G, Q103H-S248N, Q103H-S249A, Q103H-T158S, S009T-A128S-K141R-N243V, S009T-N109G-A128S-K141R, S009T-N109G-A128S-K141R-N243V-S248N-K256R, S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V, S018T-Y021N-N061S-A128S-N243V-S260P, S018T-Y021N-N061S-N109G-A128S-S260P, S018T-Y021N-N109G-A128S, S018T-Y021N-N109G-A128S-N243V, S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S033T-N109G-A128S-N243P-S248N-K256R, S033T-N243V, S033T-Q103H, S033T-T158S, S063G, S063G-A088T, S063G-A128S, S063G-K256R, S063G-L257G, S063G-N076D, S063G-N109G, S063G-Q103H, S063G-S162G, S063G-S248N, S063G-T158S, S162G, S162G-L257G, S162G-N243V, S162G-S248N, S248N, S248N-L257G, S249A, T158S, T158S-L257G, T158S-N218S, T158S-N243V, T158S-S248N, and T158S-S249A, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this BMI microswatch cleaning assay Test Method 3), the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay(test Method 3) in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-A088T, A001E-A116T, A001E-A128S-G131H-N243V, A001E-G131H-G169A-N243V, A001E-K256R, A001E-N109G, A001E-N243V, A001E-S033T, A001E-S033T-N109G-N218S, A001E-S033T-N109G-N243V, A001E-S162G, A001E-T158S, A088T-A128S, A088T-G169A, A088T-N218S, A088T-Q206D, A116T-G169A, A116T-N218S, A116T-Q206D, A128S, A128S-G131H, A128S-G169A, A128S-N218S, A128S-N243V, A128S-Q206D, G024E, G024E-A088T, G024E-A128S, G024E-G131H, G024E-K043Y, G024E-N218S, G024E-Q103H, G024E-S033T, G024E-S063G, G024E-S162G, G024E-S248N, G024E-S249A, G131H-G169A, G131H-N218S, G131H-N243V, G131H-Q206D, G169A, G169A-K256R, G169A-L257G, G169A-N218S, G169A-N243V, G169A-Q206D, G169A-S248N, G169A-S249A, K043Y, K043Y-A116T, K043Y-A128S, K043Y-G131H, K043Y-G169A, K043Y-L257G, K043Y-N109G, K043Y-N218S, K043Y-Q103H, K043Y-S063G, K043Y-S162G, K043Y-S248N, K043Y-S249A, K043Y-T158S, N076D, N076D-A088T, N076D-A128S, N076D-G131H, N076D-N218S, N076D-N243V, N076D-Q103H, N076D-S162G, N076D-S248N, N076D-S249A, N109G-G169A, N109G-Q206D, N109G-S248N, N218S, N218S-K256R, N218S-L257G, N218S-S248N, N218S-S249A, P040E-N109G-A128S-G131H, Q103H, Q103H-G169A, Q206D, Q206D-K256R, Q206D-L257G, Q206D-N218S, Q206D-N243V, Q206D-S248N, Q206D-S249A, S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T, S033T-A088T, S033T-A116T, S033T-A128S, S033T-A128S-G131H-N243P, S033T-G131H, S033T-K043Y, S033T-K256R, S033T-L257G, S033T-N076D, S033T-N076D-A128S-N218S, S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R, S033T-N109G, S033T-N109G-A128S-N243V-S248N-K256R, S033T-N218S, S033T-P040E-Q103H-N109G, S033T-Q103H-A128S-G131H, S033T-Q206D, S033T-S063G, S033T-S162G, S033T-S248N, S033T-S249A, S063G-A116T, S063G-G131H, S063G-G169A, S063G-N109G-A128S-G131H, S063G-N218S, S063G-N243V, S063G-Q206D, S063G-S249A, S162G-G169A, S162G-N218S, S162G-Q206D, S162G-S249A, S248N-K256R, S248N-S249A, S249A-K256R, S249A-L257G, T158S-G169A, T158S-K256R, T158S-Q206D, and T158S-S162G, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), and/or a PI value of 0.9 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E, A001E-A128S, A001E-G024E, A001E-G131H, A001E-G169A, A001E-L257G, A001E-N218S, A001E-Q103H, A001E-S063G, A001E-S248N, A001E-S249A, G024E-N076D, K043Y-N076D, K043Y-Q206D, N076D-A116T, N076D-G169A, N076D-Q206D, Q103H-Q206D, S033T-G169A, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, A001E-K043Y, A001E-N076D, A001E-N076D-N109G-A128S, A001E-Q206D, and G024E-Q206D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in a BMI microswatch cleaning assay (test Method 3) in Detergent Composition 4 at pH 7 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A116T, A088T-N243V, G024E-A116T, K043Y, N076D-A116T, N218S-S248N, S033T-N243V, S033T-S063G, S248N-L257G, A001E-S249A, A088T-A116T, A088T-A128S, A088T-G131H, A088T-L257G, A088T-N109G, A088T-S248N, A088T-S249A, A116T-N243V, A116T-T158S, A128S, A128S-K256R, A128S-L257G, A128S-N243V, A128S-S248N, A128S-T158S, G024E-A088T, G024E-A128S, G024E-G131H, G024E-K256R, G024E-L257G, G024E-N218S, G024E-N243V, G024E-S162G, G024E-S249A, G024E-T158S, G131H, G131H-K256R, G131H-S249A, K043Y-A088T, K043Y-A116T, K256R, N076D-K256R, N109G, N109G-A116T, N109G-A128S, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A, N109G-G131H, N109G-K256R, N109G-L257G, N109G-N218S, N109G-N243V, N109G-S248N, N218S-L257G, N243V, N243V-K256R, N243V-L257G, N243V-S248N, N243V-S249A, Q103H-A128S, Q103H-G131H, Q103H-K256R, Q103H-L257G, Q103H-N243V, Q103H-S248N, Q103H-S249A, Q103H-T158S, Q206D-N243V, S033T-A128S, S033T-K256R, S033T-N076D, S033T-N218S, S033T-S248N, S033T-T158S, S063G-A128S, S063G-K256R, S063G-N243V, S063G-S162G, S063G-T158S, S162G-K256R, S248N-K256R, S249A, T158S-N243V, and T158S-S249A, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay(Test Method 3) in Detergent Composition 4 at pH 7 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A001E-A128S, A001E-G131H, A001E-K256R, A001E-N218S, A001E-N243V, A001E-S033T, A001E-S063G, A001E-S162G, A088T, A088T-K256R, A088T-N218S, A088T-Q103H, A088T-S162G, A088T-T158S, A116T-A128S, A116T-G131H, A116T-K256R, A116T-L257G, A116T-S162G, A116T-S248N, A116T-S249A, A128S-G169A, A128S-N218S, A128S-S162G, A128S-S249A, G024E, G024E-N109G, G024E-Q103H, G024E-S033T, G024E-S063G, G024E-S248N, G131H-L257G, G131H-N243V, G131H-S162G, G131H-T158S, G169A, G169A-L257G, G169A-S248N, K043Y-A128S, K043Y-G131H, K043Y-K256R, K043Y-L257G, K043Y-N109G, K043Y-N243V, K043Y-Q103H, K043Y-S063G, K043Y-S162G, K043Y-S248N, K043Y-S249A, K043Y-T158S, K256R-L257G, L257G, N061G-N109G-N243V, N061S-A128S-N243V-S260P, N061S-N109G-A128S-N243V-S260P, N061S-N109G-A128S-S260P, N076D-A088T, N076D-A128S, N076D-G169A, N076D-N218S, N076D-N243V, N076D-S162G, N076D-S248N, N076D-T158S, N109A-A128S-N243V-K256R, N109G-A128S-G131H-N243V-S248N-K256R, N109G-A128S-N243V-S248A-K256R, N109G-A128S-N243V-S248N-K256R-L257G, N109G-A128S-S162G-N243V-S248N-K256R, N109G-A128S-T158S-N243V-S248N-K256R, N109G-Q206D, N109G-S162G, N109G-S249A, N109G-T158S, N109Q-A128S-N243V-K256R, N109S-A128S-N243V-K256R, N218S, N218S-K256R, N218S-N243V, P040A-N109G-A128S-N243V-S248N-K256R, Q103H, Q103H-A116T, Q103H-G169A, Q103H-N109G, Q103H-N218S, Q103H-S162G, S009T-A128S-K141R-N243V, S009T-N109G-A128S-K141R, S009T-N109G-A128S-K141R-N243V, S009T-S018T-Y021N-N109G-A128S-K141R, S018T-Y021N-N109G-A128S, S018T-Y021N-N109G-A128S-N243V, S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S033T-A088T, S033T-A116T, S033T-G131H, S033T-K043Y, S033T-L257G, S033T-N109G, S033T-Q103H, S033T-Q206D, S033T-S162G, S033T-S249A, S063G, S063G-A088T, S063G-A116T, S063G-L257G, S063G-N076D, S063G-N109G, S063G-N218S, S063G-Q103H, S063G-S248N, S063G-S249A, S162G, S162G-G169A, S162G-L257G, S162G-N218S, S162G-N243V, S162G-S248N, S162G-S249A, S248N, S248N-S249A, S249A-K256R, S249A-L257G, T158S, T158S-G169A, T158S-K256R, T158S-L257G, T158S-N218S, and T158S-S248N, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ protease (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 7 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E, A001E-A088T, A001E-A116T, A001E-G169A, A001E-L257G, A001E-N109G, A001E-S033T-N109G-N243V, A001E-T158S, A088T-G169A, A088T-Q206D, A116T-N218S, A128S-G131H, A128S-N243V-S248N-K256R, A128S-Q206D, G024E-K043Y, G024E-N076D, G024E-Q206D, G131H-G169A, G131H-N218S, G131H-N243V-K256R, G131H-Q206D, G131H-S248N, G169A-K256R, G169A-N218S, G169A-N243V, G169A-Q206D, K043Y-N076D, K043Y-N218S, N061P-N109G-G131H-N243V, N061P-N109G-N243V, N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-N243V, N076D, N076D-G131H, N076D-L257G, N076D-N109G, N076D-Q103H, N076D-S249A, N109G-A128S-N243V-S248N, N109G-A128S-N243V-S248N-K256R, N109G-A128S-S248N-K256R, N109G-N243P-S248A-K256R, N109G-N243P-S248N-K256R, N109G-N243V-K256R, N109G-N243V-S248A-K256R, N109G-N243V-S248N, N109G-N243V-S248N-K256R, N109G-S248N-K256R, N218S-S249A, N243V-S248N-K256R, Q103H-Q206D, Q206D, Q206D-K256R, Q206D-N218S, Q206D-S248N, Q206D-S249A, S009T-N109G-A128S-K141R-N243V-S248N-K256R, S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V, S018T-Y021N-N061S-A128S-N243V-S260P, S018T-Y021N-N061S-N109G-A128S-S260P, S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T, S033T-G169A, S033T-N076D-A128S-N218S, S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R, S033T-N109G-A128S-N243P-S248N-K256R, S033T-N109G-A128S-N243V-S248N-K256R, S063G-G131H, S063G-G169A, S162G-Q206D, and T158S-S162G, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 7 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-A128S-G131H-N243V, A001E-G024E, A001E-G131H-G169A-N243V, A001E-Q103H, A001E-S033T-N109G-N218S, A001E-S248N, A116T-G169A, A116T-Q206D, G169A-S249A, K043Y-G169A, N109G-G169A, P040E-N109G-A128S-G131H, Q206D-L257G, S033T-A128S-G131H-N243P, S033T-A128S-G131H-N243V, S033T-P040E-Q103H-N109G, S033T-Q103H-A128S-G131H, S063G-N109G-A128S-G131H, S063G-Q206D, T158S-Q206D, A001E-K043Y, A001E-N076D, A001E-Q206D, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, A001E-N076D-N109G-A128S, K043Y-Q206D, and N076D-Q206D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-L257G, G024E-K256R, G024E-L257G, N109G-A116T, N109G-L257G, N243V-K256R, S033T-N109G, S033T-T158S, S063G-L257G, A001E-L257G, A088T-A128S, A088T-G169A, A088T-K256R, A088T-N109G, A088T-N218S, A088T-N243V, A088T-S248N, A088T-T158S, A116T, A116T-A128S, A116T-G131H, A116T-K256R, A116T-L257G, A116T-N218S, A116T-S162G, A116T-T158S, A128S, A128S-G169A, A128S-K256R, A128S-L257G, A128S-N218S, G024E, G024E-A128S, G024E-G131H, G024E-N109G, G024E-N243V, G024E-S033T, G024E-S063G, G024E-S248N, G024E-S249A, G024E-T158S, G131H, G131H-G169A, G131H-K256R, G131H-N218S, G131H-S249A, G169A, G169A-L257G, G169A-N243V, K043Y-A088T, K043Y-N109G, K256R, K256R-L257G, N061G-N109G-N243V, N076D-N109G, N109G, N109G-A128S, N109G-G131H, N109G-K256R, N109G-N218S, N109G-S162G, N109G-S248N, N109G-S249A, N109G-T158S, N218S, N218S-K256R, N218S-L257G, N218S-S248N, N243V, N243V-L257G, N243V-S248N, N243V-S249A, P040A-N109G-A128S-N243V-S248N-K256R, Q103H-K256R, Q103H-L257G, Q103H-N109G, S009T-S018T-Y021N-N109G-A128S-K141R, S033T-A088T, S033T-A116T, S033T-A128S, S033T-G131H, S033T-K043Y, S033T-K256R, S033T-L257G, S033T-N076D, S033T-N218S, S033T-N243V, S033T-Q103H, S033T-S063G, S033T-S162G, S033T-S248N, S033T-S249A, S063G, S063G-A088T, S063G-A116T, S063G-A128S, S063G-G131H, S063G-K256R, S063G-N109G, S063G-N218S, S063G-N243V, S063G-S248N, S063G-S249A, S063G-T158S, S162G-K256R, S162G-N218S, S162G-N243V, S162G-S248N, S162G-S249A, S248N, S249A, S249A-L257G, T158S, T158S-L257G, and T158S-N243V, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A001E, A001E-A116T, A001E-G131H, A001E-G169A, A001E-K256R, A001E-N109G, A001E-S033T-N109G-N243V, A001E-S063G, A001E-S248N, A001E-S249A, A001E-T158S, A088T, A088T-A116T, A088T-G131H, A088T-Q103H, A088T-Q206D, A088T-S162G, A088T-S249A, A116T-G169A, A116T-N243V, A116T-S248N, A116T-S249A, A128S-G131H, A128S-N243V, A128S-S162G, A128S-S248N, A128S-S249A, A128S-T158S, G024E-A088T, G024E-A116T, G024E-K043Y, G024E-N076D, G024E-N218S, G024E-Q103H, G024E-S162G, G131H-L257G, G131H-N243V, G131H-N243V-K256R, G131H-S162G, G131H-S248N, G131H-T158S, G169A-K256R, G169A-N218S, G169A-Q206D, G169A-S248N, G169A-S249A, K043Y, K043Y-A116T, K043Y-A128S, K043Y-G169A, K043Y-K256R, K043Y-L257G, K043Y-N076D, K043Y-N218S, K043Y-N243V, K043Y-S063G, K043Y-S248N, K043Y-S249A, K043Y-T158S, L257G, N061P-N109G-G131H-N243V, N061P-N109G-N243V, N061S-A128S-N243V-S260P, N061S-N109G-A128S-N243V-S248N-K256R-S260P, N061S-N109G-A128S-S260P, N061S-N109G-N243V, N076D, N076D-A088T, N076D-A116T, N076D-G131H, N076D-G169A, N076D-K256R, N076D-L257G, N076D-N218S, N076D-N243V, N076D-Q103H, N076D-S249A, N076D-T158S, N109A-A128S-N243V-K256R, N109G-A128S-G131H-N243V-S248N-K256R, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A, N109G-A128S-N243V-S248A-K256R, N109G-A128S-N243V-S248N, N109G-A128S-N243V-S248N-K256R, N109G-A128S-N243V-S248N-K256R-L257G, N109G-A128S-S162G-N243V-S248N-K256R, N109G-G169A, N109G-N243P-S248A-K256R, N109G-N243V-K256R, N109G-N243V-S248A-K256R, N109G-N243V-S248N, N109G-N243V-S248N-K256R, N109G-S248N-K256R, N109Q-A128S-N243V-K256R, N109S-A128S-N243V-K256R, N218S-N243V, N218S-S249A, Q103H, Q103H-A116T, Q103H-A128S, Q103H-G131H, Q103H-G169A, Q103H-N218S, Q103H-N243V, Q103H-S162G, Q103H-S248N, Q103H-S249A, Q103H-T158S, Q206D, Q206D-L257G, Q206D-N218S, S009T-A128S-K141R-N243V, S009T-N109G-A128S-K141R, S009T-N109G-A128S-K141R-N243V, S009T-N109G-A128S-K141R-N243V-S248N-K256R, S009T-S018T-Y021N-A128S-K141R-N243V, S018T-Y021N-A128S-N243V, S018T-Y021N-N109G-A128S, S018T-Y021N-N109G-A128S-N243V, S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T, S033T-A128S-G131H-N243V, S033T-G169A, S033T-N109G-A128S-N243P-S248N-K256R, S033T-N109G-A128S-N243V-S248N-K256R, S033T-Q103H-A128S-G131H, S033T-Q206D, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, S063G-G169A, S063G-N076D, S063G-N109G-A128S-G131H, S063G-Q103H, S063G-S162G, S162G, S162G-G169A, S162G-L257G, S248N-K256R, S248N-L257G, S248N-S249A, S249A-K256R, T158S-G169A, T158S-K256R, T158S-N218S, T158S-S162G, T158S-S248N, and T158S-S249A, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ protease (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-A088T, A001E-A128S, A001E-A128S-G131H-N243V, A001E-G024E, A001E-G024E-S204E-Q206D, A001E-G131H-G169A-N243V, A001E-K043Y, A001E-N076D, A001E-N076D-N109G-A128S, A001E-N218S, A001E-N243V, A001E-Q103H, A001E-Q206D, A001E-S033T, A001E-S033T-N109G-N218S, A001E-S162G, A116T-Q206D, A128S-N243V-S248N-K256R, A128S-Q206D, G024E-Q206D, G131H-Q206D, K043Y-G131H, K043Y-Q103H, K043Y-Q206D, K043Y-S162G, N061S-N109G-A128S-N243V-S260P, N076D-A128S, N076D-Q206D, N076D-S162G, N076D-S248N, N109G-A128S-S248N-K256R, N109G-A128S-T158S-N243V-S248N-K256R, N109G-N243P-S248N-K256R, N109G-Q206D, N243V-S248N-K256R, P040E-N109G-A128S-G131H, Q103H-Q206D, Q206D-K256R, Q206D-N243V, Q206D-S248N, Q206D-S249A, S018T-Y021N-N061S-A128S-N243V-S260P, S018T-Y021N-N061S-N109G-A128S-S260P, S033T-A128S-G131H-N243P, S033T-N076D-A128S-N218S, S033T-N076D-N109G-A128S-N218S-N243V-S248N-K256R, S033T-P040E-Q103H-N109G, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P, S063G-Q206D, S162G-Q206D, and T158S-Q206D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in a grass microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of T158S-L257G, K256R, L257G, S033T-N109G, S162G-K256R, S162G-L257G, G024E-K256R, G024E-L257G, G024E-S033T, N109G-A116T, N218S-L257G, S033T-A088T, S033T-A116T, S033T-N243V, S033T-Q103H, S162G-N218S, S162G-N243V, T158S, T158S-N218S, T158S-N243V, A088T, A088T-G169A, A088T-K256R, A088T-L257G, A088T-S162G, A088T-T158S, A116T-K256R, A116T-L257G, A116T-N243V, A128S-L257G, A128S-N218S, A128S-N243V, A128S-S248N, G024E-A116T, G024E-A128S, G024E-G131H, G024E-N243V, G024E-S248N, G024E-S249A, G024E-T158S, G131H-N243V, G131H-T158S, G169A-N218S, G169A-N243V, G169A-S248N, K256R-L257G, N109G-A128S, N109G-G131H, N109G-N218S, N109G-N243V, N109G-S249A, N218S, N218S-K256R, N218S-N243V, N218S-S249A, N243V, N243V-K256R, N243V-L257G, N243V-S248N, Q103H-N109G, Q103H-N218S, S033T-A128S, S033T-L257G, S033T-N218S, S033T-S162G, S033T-S248N, S033T-T158S, S063G-K256R, S063G-L257G, S162G, S162G-G169A, S162G-S248N, S248N, S248N-K256R, S248N-L257G, S249A, T158S-S162G, and T158S-S248N, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in a grass microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A001E-A088T, A001E-A116T, A088T-A128S, A088T-N243V, A088T-Q103H, A088T-S248N, A088T-S249A, A116T, A116T-G169A, A116T-N218S, A116T-S162G, A116T-S249A, A116T-T158S, A128S-G169A, A128S-K256R, A128S-S162G, A128S-S249A, A128S-T158S, G024E-A088T, G024E-K043Y, G024E-N218S, G024E-Q103H, G024E-S063G, G024E-S162G, G131H-G169A, G131H-K256R, G131H-N218S, G131H-S162G, G131H-S248N, G131H-S249A, G169A, G169A-L257G, G169A-S249A, N076D, N076D-K256R, N076D-L257G, N076D-S162G, N076D-S249A, N109G-K256R, N109G-L257G, N109G-S248N, N243V-S249A, Q103H-A116T, Q103H-G169A, Q103H-K256R, Q103H-L257G, Q103H-N243V, Q103H-S162G, S033T-G131H, S033T-G169A, S033T-K043Y, S033T-N076D, S033T-Q206D, S063G, S063G-A116T, S063G-A128S, S063G-N243V, S063G-S162G, S063G-S248N, S063G-S249A, S063G-T158S, S249A-L257G, T158S-G169A, and T158S-K256R, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a grass microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-G169A, A001E-K256R, A001E-N109G, A001E-N218S, A088T-A116T, A088T-G131H, A088T-N109G, A088T-N218S, A116T-A128S, A116T-G131H, A116T-S248N, A128S, A128S-G131H, G024E, G024E-N109G, G131H, G131H-L257G, G169A-K256R, G169A-Q206D, K043Y, K043Y-A088T, K043Y-L257G, K043Y-N109G, N076D-A088T, N076D-G131H, N076D-G169A, N076D-N243V, N076D-T158S, N109G, N109G-S162G, N109G-T158S, N218S-S248N, Q103H-A128S, Q103H-S248N, Q103H-S249A, Q103H-T158S, Q206D-K256R, Q206D-L257G, Q206D-N218S, Q206D-N243V, Q206D-S248N, S033T, S033T-K256R, S033T-S063G, S033T-S249A, S063G-A088T, S063G-G131H, S063G-G169A, S063G-N109G, S162G-Q206D, S162G-S249A, S248N-S249A, S249A-K256R, T158S-Q206D, T158S-S249A, A001E-L257G, A001E-N243V, A001E-Q103H, A001E-S063G, A001E-S162G, A001E-T158S, G024E-N076D, G131H-Q206D, K043Y-A116T, K043Y-G169A, K043Y-K256R, K043Y-N076D, K043Y-S063G, K043Y-S162G, K043Y-S248N, K043Y-S249A, K043Y-T158S, N076D-A116T, N076D-A128S, N076D-S248N, N109G-G169A, Q103H, Q103H-G131H, Q206D-S249A, S063G-N076D, S063G-N218S, S063G-Q103H, A001E-A128S, A001E-G024E, A001E-G131H, A001E-N076D, A001E-Q206D, A001E-S033T, A001E-S248N, A001E-S249A, A088T-Q206D, A116T-Q206D, A128S-Q206D, G024E-Q206D, K043Y-A128S, K043Y-G131H, K043Y-N218S, K043Y-Q103H, N076D-N109G, N076D-N218S, N076D-Q103H, N109G-Q206D, Q103H-Q206D, Q206D, A001E, A001E-K043Y, K043Y-N243V, S063G-Q206D, N076D-Q206D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this grass microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an AAPF proteolytic assay: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of S033T-N076D-A128S-N218S, A001E-S033T-N109G-N218S, S033T-N218S, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243V, A128S-G169A, S033T-S063G-Q103H-N109Q-A128S-G131H-G169A-N243P, S018T-Y021N-S033T-N109G-A128S-N243V-S248N-K256R, S033T-A128S-G131H-N243P, P040E-N109G-A128S-G131H, S033T-A128S, S033T-N109G-A128S-N243V-S248N-K256R, N109G-G169A, S063G-N109G-A128S-G131H, G169A, N109G-A128S-G131H-N243V-S248N-K256R, S033T-A128S-G131H-N243V, A128S-N218S, A001E-G169A, A088T-G169A, G169A-L257G, N109G-N218S, S033T-N109G-A128S-N243P-S248N-K256R, G169A-K256R, N076D-G169A, A001E-G131H-G169A-N243V, G169A-S249A, S033T-N109G, G169A-S248N, K043Y-G169A, K043Y-N218S, N218S-L257G, N218S-N243V, S063G-G169A, A001E-A128S-G131H-N243V, A001E-S033T-N109G-N243V, A088T-N218S, G024E-N218S, G024E-S033T, G169A-Q206D, N076D-N218S, S033T-L257G, S162G-G169A, A001E-N218S, A116T-N218S, G169A-N243V, N218S, P040A-N109G-A128S-N243V-S248N-K256R, S033T-N076D, A001E-S033T, A128S-G131H, N218S-S248N, S018T-Y021N-N109G-A128S, S033T-K043Y, S033T-N243V, S033T-Q206D, S063G-N218S, S162G-N218S, T158S-G169A, A116T-G169A, G131H-G169A, N061S-N109G-A128S-S260P, N109G-A128S-N243V-K256R, N109G-A128S-N243V-S248A, N109G-A128S-N243V-S248A-K256R, N109G-A128S-N243V-S248N-K256R-L257G, N218S-K256R, S009T-N109G-A128S-K141R, S009T-S018T-Y021N-N109G-A128S-K141R, S033T-A088T, S033T-S063G, S033T-S162G, T158S-N218S, A001E-N076D-N109G-A128S, N109G-A128S-N243V-S248N-K256R, N109G-A128S-S248N-K256R, S009T-N109G-A128S-K141R-N243V, S018T-Y021N-N061S-N109G-A128S-S260P, S033T-A116T, S033T-S248N, S033T-S249A, S033T-T158S, G131H-N218S, N109A-A128S-N243V-K256R, N109G-A128S, N109G-A128S-S162G-N243V-S248N-K256R, N109G-A128S-T158S-N243V-S248N-K256R, N218S-S249A, Q206D-N218S, S018T-Y021N-N109G-A128S-N243V, S018T-Y021N-N109G-A128S-N243V-S248N-K256R, S033T-K256R, A116T-A128S, N061S-N109G-A128S-N243V-S260P, N109G-A128S-N243V-S248N, S009T-N109G-A128S-K141R-N243V-S248N-K256R, G024E-A128S, N061S-N109G-A128S-N243V-S248N-K256R-S260P, N109S-A128S-N243V-K256R, S033T, S033T-G131H, A001E-A128S, A128S, A128S-L257G, A128S-Q206D, N109Q-A128S-N243V-K256R, S009T-A128S-K141R-N243V, S009T-S018T-Y021N-A128S-K141R-N243V, A088T-A128S, A128S-K256R, A128S-N243V, N061P-N109G-N243V, N061S-A128S-N243V-S260P, S018T-Y021N-A128S-N243V, A128S-N243V-S248N-K256R, A128S-S248N, A128S-S249A, N076D-A128S, S063G-A128S, A128S-S162G, A128S-T158S, S018T-Y021N-N061S-A128S-N243V-S260P, S033T-Q103H-A128S-G131H, N061S-N109G-N243V, K043Y-A128S, N061P-N109G-G131H-N243V, N109G-L257G, A001E-G024E-S204E-Q206D, A001E-L257G, A088T-N109G, G024E-N109G, K043Y-N109G, N061G-N109G-N243V, N076D-N109G, N109G, N109G-A116T, N109G-K256R, N109G-N243V-K256R, N109G-N243V-S248A-K256R, N109G-Q206D, S063G-N109G, A001E-A116T, A001E-N109G, A001E-Q206D, A088T-A116T, A088T-N243V, A116T-L257G, G024E-A116T, G024E-L257G, G024E-N243V, G024E-Q206D, N109G-G131H, N109G-N243V, N109G-S162G, N109G-S248N, N109G-S248N-K256R, N109G-S249A, N109G-T158S, N243V-L257G, A001E-A088T, A001E-G024E, A001E-K256R, A001E-N076D, A001E-N243V, A088T, A088T-L257G, A088T-Q206D, A116T, A116T-K256R, A116T-N243V, G024E-A088T, G024E-K043Y, G024E-K256R, G024E-N076D, G024E-S162G, G024E-S248N, K043Y-A088T, K043Y-A116T, K043Y-L257G, K043Y-N243V, K043Y-Q206D, K256R-L257G, N076D-A116T, N076D-L257G, N076D-N243V, N076D-Q206D, N109G-N243V-S248N, N109G-N243V-S248N-K256R, N243V-K256R, Q206D, Q206D-L257G, Q206D-N243V, Q206D-S248N, S063G-K256R, S063G-L257G, T158S-L257G, A001E, A001E-K043Y, A001E-S162G, A001E-S248N, A001E-S249A, A001E-T158S, A088T-K256R, A088T-S162G, A088T-S248N, A088T-S249A, A116T-Q206D, A116T-S248N, A116T-S249A, G024E, G024E-G131H, G024E-S249A, G024E-T158S, G131H, G131H-K256R, G131H-L257G, K043Y-K256R, K043Y-N076D, K256R, L257G, N076D-A088T, N076D-K256R, N076D-S162G, N076D-S248N, N076D-S249A, N109G-N243P-S248A-K256R, N109G-N243P-S248N-K256R, N243V, Q206D-K256R, S033T-P040E-Q103H-N109G, S063G, S063G-A116T, S063G-Q206D, S162G-K256R, S162G-L257G, S162G-N243V, S162G-Q206D, S162G-S248N, S248N, S248N-L257G, S249A, S249A-L257G, T158S, T158S-N243V, and T158S-Q206D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in an AAPF proteolytic assay: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A001E-G131H, A001E-S063G, A088T-G131H, A088T-T158S, A116T-G131H, A116T-S162G, A116T-T158S, G024E-S063G,

G131H-N243V, G131H-N243V-K256R, G131H-Q206D, G131H-S249A, K043Y, K043Y-S063G, K043Y-S248N, K043Y-S249A, K043Y-T158S, N076D, N076D-G131H, N076D-T158S, N243V-S248N, N243V-S248N-K256R, N243V-S249A, Q103H-G169A, Q206D-S249A, S063G-N076D, S063G-N243V, S063G-S162G, S063G-S249A, S063G-T158S, S162G, S162G-S249A, S248N-K256R, S248N-S249A, S249A-K256R, T158S-K256R, T158S-S248N, and T158S-S249A, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN′-v36 in an AAPF proteolytic assay: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of G131H-S162G, G131H-S248N, G131H-T158S, K043Y-G131H, K043Y-S162G, S063G-A088T, S063G-G131H, S063G-S248N, T158S-S162G, Q103H-N218S, S033T-Q103H, and Q103H-A128S, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having proteolytic activity, enhanced proteolytic activity compared to BPN′, or a PI value greater than that of BPN′ (SEQ ID NO:2) in a BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X001E, X009T, X018T, X021N, X024G, X033T, X040A, X043Y, X061G/P/S, X063G, X076D, X088T, X103H, X109A/G/Q/S, X116T, X128S, X131H, X141R, X158S, X162G, X169A, X204E, X206D, X218S, X243P/V, X248A/N, X249A, X256R, X257G, and X260P, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2, and optionally wherein the variant comprises at least one substitution selected from the group of A001E, S009T, S018T, Y021N, S024G, S033T, P040A, K043Y, N061G/P/S, S063G, N076D, A088T, Q103H, N109A/G/Q/S, A116T, G128S, G131H, K141R, T158S, S162G, G169A, S204E, Q206D, N218S, N243P/V, S248A/N, S249A, K256R, L257G, and S260P. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Example 9 Construction and Cleaning Performance of Variants from a Combinatorial Library Based on BPN′-v36 Parent

A BPN′ combinatorial library based on the BPN′-v36 parent molecule was made by DNA 2.0 and delivered as a ligation reaction. For efficient transformation of B. subtilis, DNA from the ligation reaction mixture was amplified before transformation and transformants grown as described in Example 2. The variants were tested for cleaning performance using BMI microswatch assay in Detergent Composition 4 at 16° C. and pH 8 and egg microswatch assay in Detergent Composition 4 at 16° C. and pH 8. Protein content was determined using the TCA assay. Assays were performed as in Example 1 and Performance Indices were calculated relative to BPN′-v36 (i.e., BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q).

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-A116T-N243V-K256R-L257G, A088T-A116T-N243V-L257G, A088T-T158S-N218S-K256R, A088T-T158S-N218S-N243V-L257G, A088T-A116T-T158S-N218S-N243V-K256R-L257G, A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G, A088T-N109G-A116T-T158S-S248N-K256R-L257G, A088T-N109G-T158S-L257G, A114S-A116T-N218S-N243V-S248N-K256R-L257G, A116T-T158S-K256R, A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-T158S, A088T-N109G-A116T-G131H-L257G, A088T-N109G-A116T-T158S-N243V-S248N-L257G, A088T-N109G-N243V-L257G, A088T-N109G-N243V-S248N, A088T-N109G-T158S-N243V-L257G, A088T-N109G-T158S-N243V-S248N-L257G, A116T-T158S-S248N-L257G, Y006H-A116T-G131H-S248N, A088T-A116T-G131H-T158S-N218S-N243V, A088T-A116T-G131H-T158S-N243V, A088T-A116T-G131H-T158S-N243V-K256R-L257G, A088T-A116T-N218S-N243V-K256R-L257G, A088T-A116T-S248N-K256R-L257G, A088T-A116T-T158S-N218S-N243V, A088T-A116T-T158S-N243V-K256R-L257G, A088T-A116T-T158S-N243V-S248N-L257G, A088T-G131H-N243V-S248N-K256R-L257G, A088T-N109G-A116T-T158S-L257G, A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G, A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R, A088T-N109G-A116T-T158S-S248N-L257G, A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G, A088T-N109G-K256R, A088T-N109G-N243V-S248N-L257G, A088T-N109G-T158S-K256R, A088T-N109G-T158S-N243V, A088T-T158S-N243V-K256R-L257G, A116T, A116T-N218S-N243V-L257G-N269S, A116T-T158S-K256R-L257G, N109G-A116T-K256R-L257G, N109G-A116T-N243V, N109G-A116T-T158S-N243V-K256R-L257G, N109G-G131H-L257G, N109G-G131H-S248N-K256R-L257G, N109G-G131H-T158S-K256R-L257G, S003P-A116T-T158S-S248N-K256R, T158S-S248N-K256R, A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-S248N-K256R-L257G, A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G, A088T-A116T-S248N-L257G, A088T-A116T-T158S-N218S, A088T-A116T-T158S-N218S-K256R-L257G, A088T-A116T-T158S-N218S-L257G, A088T-G131H-N243V-L257G, A088T-G131H-T158S-S248N-L257G, A088T-L257G, A088T-N109G-A116T, A088T-N109G-A116T-G131H-N218S, A088T-N109G-A116T-G131H-N218S-S248N-L257G, A088T-N109G-A116T-G131H-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G, A088T-N109G-A116T-N218S-N243V-K256R, A088T-N109G-A116T-N218S-N243V-L257G, A088T-N109G-A116T-N243V-S248N-K256R, A088T-N109G-A116T-N243V-S248N-K256R-L257G, A088T-N109G-A116T-T158S-L257G, A088T-N109G-A116T-T158S-N243V-L257G, A088T-N109G-G131H-T158S-N243V-S248N-K256R, A088T-N109G-G131H-T158S-W241R-S248N-K256R, A088T-N109G-K256R-L257G, A088T-N109G-L257G, A088T-N109G-N243V, A088T-N109G-N243V-K256R, A088T-N109G-N243V-K256R-L257G, A088T-N109G-S248N-K256R, A088T-N109G-T158S-N218S-K256R-L257G, A088T-N109G-T158S-N218S-N243V-S248N-K256R, A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-K256R-L257G, A088T-N109G-T158S-N243V-S248N-A274D, A088T-N109G-T158S-S248N-L257G, A088T-T158S-K256R, A088T-T158S-N218S-N243V-K256R-L257G, A088T-T158S-N243V-L257G, A116T-G131H-N218S-N243V-S248N, A116T-G131H-S248N-L257G, A116T-S248N-K256R-L257G, A116T-T158S-N218S-N243V-K256R, A116T-T158S-N218S-S248N-L257G-Q271R, A116T-T158S-N243V-K256R-L257G, A116T-T158S-N243V-S248N-L257G, G131H-S248N, G131H-T158S-I234T-N243V-K256R, G131H-W241L-N243V-S248N-K256R, N109G-A116T-G131H-A137V-T158S-S248N-K256R-L257G, N109G-A116T-G131H-A151S-N218S-K256R-L257G, N109G-A116T-G131H-T158S-N218S-N243V-K256R, N109G-A116T-G131H-T158S-N218S-S248N, N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-S248N, N109G-A116T-T158S-L257G, N109G-A116T-T158S-N218S-W241R-N243V, N109G-A116T-T158S-N243V-S248N-L257G, N109G-A116T-T158S-S248N-K256R-L257G, N109G-A116T-T158S-S248N-L257G, N109G-G131H-N218S-L257G, N109G-G131H-N218S-S248N-K256R-L257G, N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T, N109G-K256R, N109G-N243V-L257G, N109G-T158S-N218S-K256R-L257G, N109G-T158S-N218S-L257G, N109G-T158S-S248N-K256R, P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E, S003F-A088T-N109G-A116T-T158S-N243V-K256R-L257G, V004A-A088T-A116T-T158S-N218S, V004A-N109G-A116T-G131H-S248N-K256R-L257G, V004L-A116T-N218S-N243V-S248N-L257G, Y006H-N109G-N218S-N243V-S248N, A001T-A116T-T158S-N243V-L257G, A088T-A116T, A088T-A116T-G131H-L257G, A088T-A116T-G131H-N218S-L257G, A088T-A116T-G131H-N218S-S248N-K256R-L257G, A088T-A116T-G131H-N218S-S248N-L257G, A088T-A116T-G131H-N243V-K256R-L257G, A088T-A116T-G131H-N243V-L257G, A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-T158S-K256R-L257G, A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S, A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T, A088T-A116T-G131H-T158S-N218S-S248N-K256R, A088T-A116T-G131H-T158S-N218S-S248N-L257G, A088T-A116T-G131H-T158S-N243V-S248N-K256R, A088T-A116T-G131H-T158S-S248N, A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-K256R, A088T-A116T-K256R-L257G, A088T-A116T-N218S-N243V-L257G, A088T-A116T-N243V-K256R, A088T-A116T-N243V-L257G, A088T-A116T-N243V-S248N-K256R-L257G, A088T-A116T-S248N-K256R, A088T-A116T-T158S-K256R, A088T-A116T-T158S-N218S, A088T-A116T-T158S-N218S-N243V-K256R, A088T-A116T-T158S-N218S-N243V-K256R-N269S, A088T-A116T-T158S-N218S-N243V-S248N, A088T-A116T-T158S-N218S-N243V-S248N, A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-A116T-T158S-N243V-K256R, A088T-A116T-T158S-N243V-L257G, A088T-A116T-T158S-N243V-S248N-K256R, A088T-A116T-T158S-N243V-S248N-K256R-L257G, A088T-A116T-T158S-S248N-K256R, A088T-A116T-V143A-N218S-S248N-K256R, A088T-A116T-V147I-T158S-N218S-N243V-L257G, A088T-G131H-K256R-L257G, A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-S248N-L257G, A088T-G131H-S248N-K256R-L257G, A088T-G131H-T158S-L257G, A088T-G131H-T158S-N218S-K256R, A088T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-G131H-T158S-N218S-N243V-L257G, A088T-G131H-T158S-N218S-S248N, A088T-G131H-T158S-N243V, A088T-G131H-T158S-N243V, A088T-G131H-T158S-N243V-S248N, A088T-G131H-T158S-N243V-S248N-K256R, A088T-G131H-T158S-N243V-S248N-L257G, A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N, A088T-N109G-A116T-G131H-L257G, A088T-N109G-A116T-G131H-N218S, A088T-N109G-A116T-G131H-N218S-L257G, A088T-N109G-A116T-G131H-N218S-N243V, A088T-N109G-A116T-G131H-N218S-N243V-K256R-L257G, A088T-N109G-A116T-G131H-N218S-N243V-L257G, A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-N243V, A088T-N109G-A116T-G131H-N243V-L257G, A088T-N109G-A116T-G131H-N243V-S248N-L257G, A088T-N109G-A116T-G131H-S248N, A088T-N109G-A116T-G131H-S248N-K256R, A088T-N109G-A116T-G131H-S248N-L257G, A088T-N109G-A116T-G131H-T158S-L257G, A088T-N109G-A116T-G131H-T158S-N218S, A088T-N109G-A116T-G131H-T158S-N218S-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218T-N243V, A088T-N109G-A116T-G131H-T158S-N243V-K256R, A088T-N109G-A116T-G131H-T158S-N243V-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N243V-S248N, A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-S248N-L257G, A088T-N109G-A116T-N218S, A088T-N109G-A116T-N218S-L257G, A088T-N109G-A116T-N218S-N243V, A088T-N109G-A116T-N218S-N243V-S248N-L257G, A088T-N109G-A116T-N218S-S248N-K256R, A088T-N109G-A116T-N218T-K256R, A088T-N109G-K256R, A088T-N109G-A116T-N218T-K256R, A088T-N109G-A116T-N218T-K256R-L257G, A088T-N109G-A116T-N243V, A088T-N109G-A116T-N243V-K256R-L257G, A088T-N109G-A116T-N243V-K256R-L257G-N269D, A088T-N109G-A116T-S248N-K256R, A088T-N109G-A116T-T158S, A088T-N109G-A116T-T158S-N218S-L257G, A088T-N109G-A116T-T158S-N218S-N243V, A088T-N109G-A116T-T158S-N218S-N243V-K256R, A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G, A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G, A088T-N109G-A116T-T158S-N218S-N243V-L257G, A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-A116T-T158S-N243V, A088T-N109G-A116T-T158S-N243V-K256R, A088T-N109G-A116T-T158S-N243V-K256R-L257G, A088T-N109G-A116T-T158S-S248N-L257G, A088T-N109G-G131H-L257G, A088T-N109G-G131H-N218S-K256R-L257G, A088T-N109G-G131H-N218S-N243V-K256R, A088T-N109G-G131H-N218S-N243V-L257G, A088T-N109G-G131H-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-N243V, A088T-N109G-G131H-N243V-L257G, A088T-N109G-G131H-N243V-S248N-K256R, A088T-N109G-G131H-N243V-S248N-L257G, A088T-N109G-G131H-S248N-L257G, A088T-N109G-G131H-T158S-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R, A088T-N109G-G131H-T158S-N243V, A088T-N109G-G131H-T158S-N243V-K256R, A088T-N109G-G131H-T158S-N243V-K256R-L257G, A088T-N109G-G131H-T158S-N243V-L257G, A088T-N109G-L257G, A088T-N109G-N218S-K256R, A088T-N109G-N218S-N243V-S248N-L257G, A088T-N109G-N218S-S248N-K256R-L257G, A088T-N109G-N243V-K256R-L257G, A088T-N109G-N243V-S248N-K256R-L257G, A088T-N109G-N243V-S248N-L257G-I268V, A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S-N218S-K256R, A088T-N109G-T158S-N218S-N243V-L257G, A088T-N109G-T158S-N218S-N243V-L257G, A088T-N109G-T158S-N243V-K256R-I268V, A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N218S-N243V, A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-S248N, A088T-N218S-S248N-L257G, A088T-N243V, A088T-N243V, A088T-N243V-K256R, A088T-N243V-L257G, A088T-S145T-T158S-S248N, A088T-T158S-L257G, A088T-T158S-N218S-S248N-L257G, A088T-T158S-N243V-K256R-L257G-Q271H, A088T-T158S-S248N, A088T-V143A-T158S-K256R, A116T-G131H-K256R, A116T-G131H-N218S, A116T-G131H-N243V, A116T-G131H-N243V-K256R, A116T-G131H-N243V-L257G, A116T-G131H-S248N-K256R, A116T-G131H-T158S-N218S-I234T-N243V-S248N-K256R, A116T-G131H-T158S-N243V-L257G, A116T-G131H-T158S-N243V-S248N-K256R, A116T-G131H-V143F-T158S-N218S, A116T-L257G, A116T-N218S, A116T-N218S-L257G, A116T-N218S-N243V-L257G, A116T-N243V, A116T-N243V-K256R, A116T-N243V-S248N, A116T-N243V-S248N-K256R-L257G, A116T-S248N, A116T-T158S, A116T-T158S-N218S-N243V, A116T-T158S-N218S-S248N, A116T-T158S-N243V, A116T-T158S-N243V-K256R, A116T-T158S-N243V-L257G, A116T-T158S-N243V-S248N, A116T-T158S-S248N-K256R-L257G, A116T-V149I-T158S-N243V-S248N-K256R-Q271H, G131H-N218S-N243V-L257G, G131H-N243V, G131H-N243V-S248N-K256R, G131H-T158S, G131H-T158S-N218S-N243V-K256R, G131H-T158S-N243V-K256R-L257G, G131H-T158S-N243V-S248N-L257G, N109G-A116T-G131H-N218S-K256R-L257G, N109G-A116T-G131H-N218S-L257G, N109G-A116T-G131H-N218S-N243V-K256R-L257G, N109G-A116T-G131H-N218S-S248N-K256R, N109G-A116T-G131H-N243V-K256R, N109G-A116T-G131H-N243V-L257G, N109G-A116T-G131H-N243V-S248N-K256R-L257G, N109G-A116T-G131H-S248N, N109G-A116T-G131H-S248N-I268V, N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R, N109G-A116T-G131H-T158S-N218S-S248N-L257G, N109G-A116T-G131H-T158S-S248N, N109G-A116T-G131H-T158S-S248N-K256R, N109G-A116T-N218S, N109G-A116T-N218S-N243V-K256R, N109G-A116T-N218S-N243V-K256R-L257G, N109G-A116T-N218S-S248N-L257G, N109G-A116T-N243V-K256R, N109G-A116T-N243V-S248N-K256R-L257G, N109G-A116T-S248N-L257G, N109G-A116T-T158S-G211V-N243V-S248N-K256R, N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S, N109G-A116T-T158S-N218S-N243V-K256R-L257G, N109G-A116T-T158S-N218S-N243V-L257G, N109G-A116T-T158S-N218S-N243V-S248N-L257G, N109G-A116T-T158S-N218S-S248N-K256R-L257G, N109G-A116T-T158S-N243V, N109G-A116T-T158S-Q275R, N109G-G131H-A137V-T158S-N218S-S248N, N109G-G131H-N218S-K237N, N109G-G131H-N218S-N243V-K256R-L257G, N109G-G131H-N218S-S248N-K256R, N109G-G131H-N243V-K256R-L257G, N109G-G131H-S145F-N218S-N243V-K256R-L257G, N109G-G131H-S248N-K256R, N109G-G131H-S248N-L257G, N109G-G131H-T158S-K256R, N109G-G131H-T158S-N218S-N243V-K256R, N109G-G131H-T158S-N243V, N109G-G131H-T158S-N243V-K256R-L257G, N109G-G131H-T158S-N243V-L257G, N109G-G131H-T158S-S248N-L257G, N109G-G131H-T158S-S248N-Q271R, N109G-N218S-L257G, N109G-N218S-N243V, N109G-N243V-K256R-L257G, N109G-N243V-S248N-K256R-L257G, N109G-T158S-I268V, N109G-T158S-K256R, N109G-T158S-N218S-N243V-K256R-L257G, N109G-T158S-N218S-S248N-L257G, N109G-T158S-N243V, N109G-T158S-N243V-K256R-L257G, N109G-T158S-N243V-S248N, N109S-A116T-S248N, N218S, N218S-N243V-S248N-K256R-L257G, N218S-S248N-L257G, N243V, N243V-K256R, N243V-S248N-K256R, N243V-S248N-K256R-L257G, S105P-A116T-T158S-N218S-N243V-S248N-K256R, S248N, T158S-N243V-K256R, and T158S-N243V-L257G, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A088T, A088T-A116T-G131H-L257G, A088T-A116T-G131H-N218S-A274T, A088T-A116T-G131H-N218S-K256R, A088T-A116T-G131H-N218S-K256R-L257G, A088T-A116T-G131H-N218S-N243V, A088T-A116T-G131H-N218S-N243V-L257G, A088T-A116T-G131H-N218S-N243V-S248N-K256R, A088T-A116T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-N218S-N243V-S248N-L257G, A088T-A116T-G131H-N218S-S248N, A088T-A116T-G131H-N218S-S248N-L257G, A088T-A116T-G131H-N243V, A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-N243V-S248N-A274V, A088T-A116T-G131H-N243V-S248N-K256R-L257G, A088T-A116T-G131H-N243V-S248N-K256R-L257G, A088T-A116T-G131H-N243V-S248N-L257G, A088T-A116T-G131H-S248N-K256R, A088T-A116T-G131H-S248N-K256R-L257G, A088T-A116T-G131H-T158S-K256R, A088T-A116T-G131H-T158S-K256R-L257G, A088T-A116T-G131H-T158S-N218S-K256R, A088T-A116T-G131H-T158S-N218S-K256R-L257G, A088T-A116T-G131H-T158S-N218S-L257G, A088T-A116T-G131H-T158S-N218S-N243V-K256R, A088T-A116T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-A116T-G131H-T158S-N218S-N243V-L257G, A088T-A116T-G131H-T158S-N218S-N243V-S248N, A088T-A116T-G131H-T158S-N218S-N243V-S248N, A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-A116T-G131H-T158S-N243V-K256R, A088T-A116T-G131H-T158S-N243V-K256R, A088T-A116T-G131H-T158S-N243V-K256R-L257G, A088T-A116T-G131H-T158S-N243V-L257G, A088T-A116T-G131H-T158S-N243V-S248N, A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-T158S-N243V-S248N-L257G, A088T-A116T-G131H-T158S-S248N-K256R, A088T-A116T-G131H-T158S-S248N-K256R-L257G, A088T-A116T-K256R, A088T-A116T-L257G, A088T-A116T-N218S, A088T-A116T-N218S-N243V-K256R, A088T-A116T-N218S-N243V-N269D, A088T-A116T-N218S-N243V-S248N, A088T-A116T-N218S-N243V-S248N-K256R-L257G, A088T-A116T-N218S-N243V-S248N-L257G, A088T-A116T-N218S-S248N, A088T-A116T-N218S-S248N-L257G, A088T-A116T-N243V-K256R-L257G, A088T-A116T-N243V-S248N-K256R, A088T-A116T-S248N, A088T-A116T-S248N-K256R, A088T-A116T-T158S-A216S-N218S-N243V-K256R-L257G, A088T-A116T-T158S-N218S-K256R, A088T-A116T-T158S-N218S-N243V-K256R, A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N243V-K256R, A088T-A116T-T158S-N243V-S248N, A088T-A116T-T158S-N243V-S248N-K256R, A088T-A116T-T158S-N243V-S248N-L257G, A088T-A116T-T158S-S248N, A088T-G131D-T158S-N243V-S248N, A088T-G131H-A138V-N218S-L257G, A088T-G131H-K256R, A088T-G131H-N218S-N243V-K256R, A088T-G131H-N218S-N243V-K256R, A088T-G131H-N218S-S248N, A088T-G131H-N218S-S248N-K256R-L257G, A088T-G131H-N218T-L257G, A088T-G131H-N243V-L257G, A088T-G131H-N243V-S248N-K256R, A088T-G131H-S248N, A088T-G131H-S248N-L257G, A088T-G131H-T158S-K256R, A088T-G131H-T158S-K256R-L257G, A088T-G131H-T158S-N218S, A088T-G131H-T158S-N218S, A088T-G131H-T158S-N218S-K256R-L257G, A088T-G131H-T158S-N218S-N243V-K256R, A088T-G131H-T158S-N218S-N243V-K256R, A088T-G131H-T158S-N218S-N243V-S248N, A088T-G131H-T158S-N218S-S248N, A088T-G131H-T158S-N218S-S248N-K256R, A088T-G131H-T158S-N218S-S248N-L257G-I268V, A088T-G131H-T158S-N243V-K256R, A088T-G131H-T158S-N243V-K256R-L257G, A088T-G131H-T158S-N243V-S248N, A088T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-G131H-T158S-S248N, A088T-G131H-T158S-S248N-K256R, A088T-N109G-A116T-G131H-K256R, A088T-N109G-A116T-G131H-K256R-L257G, A088T-N109G-A116T-G131H-K256R-L257G, A088T-N109G-A116T-G131H-N218S-K256R, A088T-N109G-A116T-G131H-N218S-K256R, A088T-N109G-A116T-G131H-N218S-K256R-L257G, A088T-N109G-A116T-G131H-N218S-N243V-L257G, A088T-N109G-A116T-G131H-N218S-S248N, A088T-N109G-A116T-G131H-N218S-S248N-L257G, A088T-N109G-A116T-G131H-N243V-K256R, A088T-N109G-A116T-G131H-N243V-K256R-L257G, A088T-N109G-A116T-G131H-N243V-S248N-K256R, A088T-N109G-A116T-G131H-N243V-S248N-K256R, A088T-N109G-A116T-G131H-S248N-K256R-L257G, A088T-N109G-A116T-G131H-S248N-K256R-L257G, A088T-N109G-A116T-G131H-S248N-L257G, A088T-N109G-A116T-G131H-T158S-K256R, A088T-N109G-A116T-G131H-T158S-L257G, A088T-N109G-A116T-G131H-T158S-N218S, A088T-N109G-A116T-G131H-T158S-N218S-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243V, A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N218S-S248N, A088T-N109G-A116T-G131H-T158S-N218T-K256R, A088T-N109G-A116T-G131H-T158S-N243V, A088T-N109G-A116T-G131H-T158S-N243V-S248N, A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G, A088T-N109G-A116T-G131H-V149A-T158S-N218S-K256R, A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R, A088T-N109G-A116T-N218S-N243V, A088T-N109G-A116T-N218S-N243V-K256R-L257G, A088T-N109G-A116T-N218S-N243V-L257G, A088T-N109G-A116T-N218S-N243V-S248N-K256R, A088T-N109G-A116T-N218S-S248N, A088T-N109G-A116T-N218S-S248N-K256R-L257G, A088T-N109G-A116T-N243V-K256R, A088T-N109G-A116T-N243V-S248N-K256R-L257G, A088T-N109G-A116T-N243V-S248N-L257G, A088T-N109G-A116T-N243V-S248N-L257G, A088T-N109G-A116T-S248N, A088T-N109G-A116T-S248N-K256R-L257G, A088T-N109G-A116T-S248N-L257G, A088T-N109G-A116T-T158S, A088T-N109G-A116T-T158S-K256R, A088T-N109G-A116T-T158S-N218S, A088T-N109G-A116T-T158S-N218S-L257G, A088T-N109G-A116T-T158S-N218S-N243V-S248N, A088T-N109G-A116T-T158S-N218S-N243V-S248N, A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R, A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-A116T-T158S-N218S-S248N-K256R, A088T-N109G-A116T-T158S-N218S-S248N-L257G, A088T-N109G-A116T-T158S-N218S-S248N-L257G, A088T-N109G-A116T-T158S-N243V-K256R, A088T-N109G-A116T-T158S-N243V-S248N, A088T-N109G-A116T-T158S-S248N, A088T-N109G-A116T-T158S-S248N-K256R, A088T-N109G-A137E-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-A152S-T158S-N218S-S248N-K256R, A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S, A088T-N109G-G131H-N218S, A088T-N109G-G131H-N218S-K256R, A088T-N109G-G131H-N218S-K256R, A088T-N109G-G131H-N218S-L257G, A088T-N109G-G131H-N218S-N243V, A088T-N109G-G131H-N218S-N243V-K256R, A088T-N109G-G131H-N218S-N243V-K256R-L257G, A088T-N109G-G131H-N218S-N243V-S248N-K256R, A088T-N109G-G131H-N218S-S248N, A088T-N109G-G131H-N218S-S248N-K256R, A088T-N109G-G131H-N218S-S248N-K256R, A088T-N109G-G131H-N218S-S248N-K256R-L257G, A088T-N109G-G131H-N243V-K256R, A088T-N109G-G131H-N243V-K256R-L257G, A088T-N109G-G131H-N243V-K256R-L257G, A088T-N109G-G131H-N243V-L257G, A088T-N109G-G131H-N243V-S248N-K256R, A088T-N109G-G131H-S248N-K256R, A088T-N109G-G131H-S248N-L257G, A088T-N109G-G131H-T158S, A088T-N109G-G131H-T158S-K256R, A088T-N109G-G131H-T158S-K256R-L257G, A088T-N109G-G131H-T158S-N218S-K256R, A088T-N109G-G131H-T158S-N218S-K256R, A088T-N109G-G131H-T158S-N218S-L257G, A088T-N109G-G131H-T158S-N218S-L257G, A088T-N109G-G131H-T158S-N218S-N243V, A088T-N109G-G131H-T158S-N218S-N243V-K256R, A088T-N109G-G131H-T158S-N218S-N243V-S248N, A088T-N109G-G131H-T158S-N218S-S248N-L257G, A088T-N109G-G131H-T158S-N243V-K256R-L257G, A088T-N109G-G131H-T158S-N243V-S248N, A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-N243V-S248N-L257G, A088T-N109G-G131H-T158S-N243V-S248N-L257G, A088T-N109G-G131H-T158S-S248N, A088T-N109G-G131H-T158S-S248N-L257G, A088T-N109G-G131H-V149A-K256R-L257G, A088T-N109G-G154A-N155P-E156T-G157L-T158M-S159E-G160E-S161L, A088T-N109G-K256R-L257G, A088T-N109G-N218S-K256R, A088T-N109G-N218S-N243V-L257G, A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-S248N, A088T-N109G-N218S-S248N-L257G, A088T-N109G-N218S-S248N-L257G, A088T-N109G-N243V-S248N-K256R, A088T-N109G-S248N, A088T-N109G-S248N, A088T-N109G-T158S-N218S, A088T-N109G-T158S-N218S-K256R-Q271H, A088T-N109G-T158S-N218S-N243V, A088T-N109G-T158S-N218S-N243V-K256R-Q275R, A088T-N109G-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-T158S-N218S-S248N, A088T-N109G-T158S-N218S-S248N-K256R, A088T-N109G-T158S-N218S-S248N-N269D, A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-S248N-K256R, A088T-N109G-T158S-N243V-S248N-K256R-L257G-N269D, A088T-N109G-T158S-N243V-S248N-L257G, A088T-N109G-T158S-S248N-K256R-L257G, A088T-N109G-T158S-S248N-L257G, A088T-N109G-V147A-N218S-N243V-K256R, A088T-N218S, A088T-N218S-K256R, A088T-N218S-L257G-I268V, A088T-N218S-N243V, A088T-N218S-N243V-K256R, A088T-N218S-N243V-K256R-L257G, A088T-N218S-N243V-L257G, A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-N243V-S248N-L257G, A088T-N218S-N243V-S248N-N269S, A088T-N218S-S248N-K256R, A088T-N243V-S248N, A088T-N243V-S248N-K256R, A088T-N243V-S248N-K256R, A088T-N243V-S248N-K256R-L257G, A088T-N243V-S248N-L257G, A088T-S248N, A088T-S248N, A088T-S248N-K256R-L257G, A088T-S248N-L257G, A088T-S248N-L257G-I268V, A088T-T158S, A088T-T158S, A088T-T158S-N218S, A088T-T158S-N218S-K256R, A088T-T158S-N218S-L257G, A088T-T158S-N218S-N243V-K256R-I268V, A088T-T158S-N218S-N243V-K256R-L257G, A088T-T158S-N218S-N243V-S248N-L257G, A088T-T158S-N218S-S248N, A088T-T158S-N243V-K256R, A088T-T158S-N243V-K256R-L257G, A088T-T158S-N243V-S248N-K256R, A088T-T158S-N243V-S248N-L257G, A088T-T158S-N243V-S248N-L257G, A088T-T158S-S248N, A088T-T158S-S248N-L257G, A088T-V147A-K256R, A098S-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A116T-G131H-N218S-K256R, A116T-G131H-N218S-K256R-L257G, A116T-G131H-N218S-L257G, A116T-G131H-N218S-N243V-S248N-L257G, A116T-G131H-N218S-S248N-K256R-L257G, A116T-G131H-N243V-S248N, A116T-G131H-N243V-S248N-L257G, A116T-G131H-T158S-A231V-N243V-L257G, A116T-G131H-T158S-K256R, A116T-G131H-T158S-K256R-L257G, A116T-G131H-T158S-N218S-K256R, A116T-G131H-T158S-N218S-K256R-L257G, A116T-G131H-T158S-N218S-N243V, A116T-G131H-T158S-N218S-N243V-K256R, A116T-G131H-T158S-N218S-N243V-K256R-L257G, A116T-G131H-T158S-N218S-N243V-L257G, A116T-G131H-T158S-N218S-N243V-S248N-K256R, A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A116T-G131H-T158S-N218S-N243V-S248N-L257G, A116T-G131H-T158S-N218S-S248N-K256R, A116T-G131H-T158S-N218T-L257G, A116T-G131H-T158S-S248N-K256R, A116T-G131H-T158S-S248N-L257G, A116T-N218S-K256R, A116T-N218S-K256R-L257G, A116T-N218S-N243V-S248N-K256R, A116T-N218S-N243V-S248N-K256R-L257G, A116T-N218S-N243V-S248N-L257G, A116T-N218S-S248N, A116T-N218S-S248N-K256R, A116T-N218S-S248N-L257G, A116T-N243V-S248N-L257G, A116T-S248N-L257G, A116T-T158S-L257G-Q271R, A116T-T158S-N218S-L257G, A116T-T158S-N218S-N243V-K256R-L257G, A116T-T158S-N218S-S248N-K256R, A116T-T158S-N218S-S248N-K256R-L257G, A116T-T158S-N243V-S248N-K256R, A116T-T158S-N243V-S248N-K256R-L257G, G024S-G053S-N078S-G097A-N101S-A128S, G131H, G131H-N218S, G131H-N218S-K256R, G131H-N218S-N243V-K256R, G131H-N218S-N243V-K256R-L257G, G131H-N218S-N243V-S248N, G131H-N218S-N243V-S248N-K256R, G131H-N218S-S248N-K256R-L257G, G131H-N218S-S248N-L257G, G131H-N243V-K256R, G131H-N243V-K256R-L257G, G131H-N243V-S248N-K256R-L257G, G131H-S248N-K256R, G131H-T158S-K256R, G131H-T158S-K256R-L257G, G131H-T158S-N218S-K256R, G131H-T158S-N218S-K256R-L257G, G131H-T158S-N218S-N243V-K256R-L257G, G131H-T158S-N218S-N243V-S248N, G131H-T158S-N218S-S248N-K256R-L257G, G131H-T158S-N218S-S248N-L257G, G131H-T158S-N243V-K256R, G131H-T158S-N243V-S248N-K256R, G131H-T158S-S248N-L257G, G131H-V147I-N218S-S248N-K256R, I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, I107T-N109G-G131H-N218S-N243V-K256R-L257G, N109G, N109G-A116T-G131H, N109G-A116T-G131H-A144V-T158S-S248N-K256R-L257G, N109G-A116T-G131H-K256R-L257G, N109G-A116T-G131H-L257G, N109G-A116T-G131H-N218S-N243V-K256R, N109G-A116T-G131H-N218S-N243V-S248N-K256R, N109G-A116T-G131H-N243V, N109G-A116T-G131H-N243V-K256R-L257G, N109G-A116T-G131H-N243V-S248N, N109G-A116T-G131H-N243V-S248N-K256R, N109G-A116T-G131H-S248N-K256R, N109G-A116T-G131H-T158S-K256R, N109G-A116T-G131H-T158S-K256R-L257G, N109G-A116T-G131H-T158S-L257G, N109G-A116T-G131H-T158S-N218S, N109G-A116T-G131H-T158S-N218S-K256R-L257G, N109G-A116T-G131H-T158S-N218S-L257G, N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, N109G-A116T-G131H-T158S-N218S-S248N-K256R, N109G-A116T-G131H-T158S-N218S-S248N-K256R-L257G, N109G-A116T-G131H-T158S-N243V-L257G, N109G-A116T-G131H-T158S-S248N-L257G, N109G-A116T-G131H-V147A-T158S-N218S-K256R-L257G, N109G-A116T-G131H-V149A-T158S-N218S-N243V-S248N-L257G, N109G-A116T-K256R, N109G-A116T-N218S-K256R, N109G-A116T-N218S-N243V, N109G-A116T-N218S-N243V-L257G, N109G-A116T-N218S-N243V-S248N-I268V, N109G-A116T-N218S-N243V-S248N-K256R-L257G, N109G-A116T-N218S-N243V-S248N-L257G, N109G-A116T-N243V-S248N, N109G-A116T-N243V-S248N-K256R, N109G-A116T-N243V-S248N-L257G, N109G-A116T-S248N-K256R, N109G-A116T-T158S, N109G-A116T-T158S-N218S-N243V-S248N-K256R, N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G, N109G-A116T-T158S-N218S-S248N-K256R, N109G-A116T-T158S-N243V-L257G, N109G-A116T-T158S-N243V-S248N, N109G-A116T-T158S-S248N, N109G-A116T-T158S-S248N-K256R, N109G-G131H, N109G-G131H-K256R, N109G-G131H-N218S-K256R, N109G-G131H-N218S-K256R-L257G, N109G-G131H-N218S-N243V-L257G, N109G-G131H-N218S-N243V-S248N-K256R, N109G-G131H-N218S-N243V-S248N-K256R-L257G, N109G-G131H-N218S-N243V-S248N-L257G, N109G-G131H-N218S-S248N-L257G, N109G-G131H-N243V, N109G-G131H-N243V-K256R, N109G-G131H-N243V-S248N, N109G-G131H-N243V-S248N-K256R-L257G, N109G-G131H-N243V-S248N-L257G, N109G-G131H-T158S-N218S-K256R-L257G, N109G-G131H-T158S-N218S-L257G, N109G-G131H-T158S-N218S-N243V, N109G-G131H-T158S-N218S-N243V-K256R-L257G, N109G-G131H-T158S-N218S-N243V-S248N, N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G, N109G-G131H-T158S-N218S-N243V-S248N-L257G, N109G-G131H-T158S-N218S-S248N-K256R-L257G, N109G-G131H-T158S-N243V-K256R-I268V, N109G-G131H-T158S-N243V-S248N, N109G-G131H-T158S-N243V-S248N-K256R, N109G-G131H-T158S-N243V-S248N-L257G, N109G-G131H-T158S-S248N, N109G-G131H-T158S-S248N-K256R-L257G, N109G-K141E-N218S-S248N-L257G, N109G-N218S, N109G-N218S-N243V-K256R, N109G-N218S-N243V-L257G, N109G-N218S-N243V-S248N-S260F, N109G-N218S-S248N, N109G-N218S-S248N-K256R, N109G-N243V-K256R, N109G-N243V-S248N, N109G-N243V-S248N-K256R, N109G-N243V-S248N-L257G, N109G-N243V-S248N-L257G-Q275R, N109G-S182F-S204F-S207L-N218S-S236F-S248N-L257G, N109G-S248N-K256R, N109G-T158S-K256R-L257G, N109G-T158S-L257G, N109G-T158S-N218S-N243V-K256R, N109G-T158S-N218S-N243V-S248N, N109G-T158S-N218S-N243V-S248N-L257G, N109G-T158S-N243V-K256R, N109G-T158S-N243V-S248N-K256R, N109G-T158S-N243V-S248N-L257G, N109G-T158S-S248N-L257G, N218S-N243V-L257G, N218S-N243V-S248N-K256R, N243V-K256R-L257G, N243V-S248N-L257G-Q271R, P057Q-A088T-N109G-A116T-G131H-T158S-N218S-S248N, S003P-A116T-N218S-K256R, S003P-N109G-G131H-N218S-N243V-S248N-K256R-L257G, S248N-K256R-L257G, T158S-K256R-L257G, T158S-N218S-A272V, T158S-N218S-K256R-L257G, T158S-N218S-L233S, T158S-N218S-N243V, T158S-N218S-N243V-K256R-L257G, T158S-N218S-N243V-L257G, T158S-N218S-N243V-S248N-K256R, T158S-N218S-S248N-K256R, T158S-N243V, T158S-N243V-K256R-L257G, T158S-N243V-S248N, T158S-N243V-S248N-K256R-N269D, and V004A-N109G-A116T-T158S-N218S-S248N-L257G, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-A098S-N218S-K256R, A088T-A116T-G131H-K256R, A088T-A116T-G131H-K256R-L257G-L267M, A088T-A116T-G131H-N218S-N243V-K256R, A088T-A116T-G131H-N218S-N243V-K256R-L257G, A088T-A116T-G131H-N218S-N243V-S248N, A088T-A116T-G131H-N218S-S248N, A088T-A116T-G131H-N218S-S248N-K256R, A088T-A116T-G131H-N218S-S248N-K256R, A088T-A116T-G131H-N243V, A088T-A116T-G131H-S248N, A088T-A116T-G131H-S248N-L257G, A088T-A116T-G131H-S248N-L257G, A088T-A116T-G131H-T158S-N218S, A088T-A116T-G131H-T158S-N218S-K256R-L257G, A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-A116T-G131H-T158S-N218S-S248N, A088T-A116T-G131H-T158S-N218S-S248N-K256R, A088T-A116T-K256R-L257G, A088T-A116T-N218S-I268V, A088T-A116T-N218S-K256R, A088T-A116T-N218S-N243V-Q271R, A088T-A116T-N218S-N243V-S248N-K256R, A088T-A116T-N218S-N243V-S248N-K256R-Q275R, A088T-A116T-N218S-S248N, A088T-A116T-N218S-S248N-K256R, A088T-A116T-N243V-S248N-K256R, A088T-A116T-T158S, A088T-A116T-T158S-N218S-K256R, A088T-A116T-T158S-N218S-N243V-L257G, A088T-A116T-T158S-N218S-N243V-S248N-L257G, A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N218S-S248N-K256R, A088T-A116T-T158S-N218S-S248N-K256R-L257G, A088T-A116T-T158S-N218S-S248N-L257G, A088T-A116T-T158S-S248N-L257G, A088T-G131H, A088T-G131H, A088T-G131H-N218S-K237R-K256R-L257G, A088T-G131H-N218S-K256R, A088T-G131H-N218S-K256R-L257G, A088T-G131H-N218S-N243V-K256R-L257G, A088T-G131H-N218S-N243V-L257G, A088T-G131H-N218S-N243V-L257G, A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-N243V-S248N-K256R, A088T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-G131H-N218S-S248N, A088T-G131H-N243V, A088T-G131H-N243V-K256R, A088T-G131H-N243V-K256R-L257G, A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N-K256R, A088T-G131H-S248N, A088T-G131H-S248N-K256R, A088T-G131H-T158S-N218S-K256R-L257G, A088T-G131H-T158S-N218S-L257G, A088T-G131H-T158S-N218S-N243V, A088T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-G131H-T158S-N218S-S248N-K256R, A088T-G131H-T158S-N218S-S248N-K256R-L257G, A088T-G131H-T158S-S248N-K256R-L257G, A088T-L257G, A088T-N109G-A116T-G131H-N218S-L257G, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-N218S-N243V-S248N-N269D, A088T-N109G-A116T-G131H-N218S-N243V-S248N-Q275R, A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-N243V-S248N, A088T-N109G-A116T-G131H-T158S, A088T-N109G-A116T-G131H-T158S-N218S-L257G-I268V, A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G, A088T-N109G-A116T-G131H-T158S-S248N, A088T-N109G-A116T-G131H-T158S-S248N, A088T-N109G-A116T-G131H-W241L-S248N-K256R-L257G, A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R-L257G, A088T-N109G-A116T-N218S-N243V-S248N-K256R, A088T-N109G-A116T-N218S-S248N, A088T-N109G-A116T-T158S-N218S, A088T-N109G-A116T-T158S-N218S-K256R-L257G, A088T-N109G-A116T-T158S-N218S-N243V-S248N-L257G, A088T-N109G-A116T-T158S-N243V-S248N, A088T-N109G-A116T-T158S-N243V-S248N-K256R, A088T-N109G-G131H-A138V-T158S-N218S-N243V-S248N-L257G, A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S-N243V, A088T-N109G-G131H-N218S-N243V-S248N-L257G, A088T-N109G-G131H-N218S-S248N-K256R-L257G, A088T-N109G-G131H-N218S-S248N-K256R-L257G-Q275R, A088T-N109G-G131H-N243V-S248N, A088T-N109G-G131H-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S, A088T-N109G-G131H-T158S-L233S-N243V-S248N, A088T-N109G-G131H-T158S-N218S-N243V-K256R, A088T-N109G-G131H-T158S-N218S-N243V-K256R-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N-L257G, A088T-N109G-G131H-T158S-N218S-S248N-K256R, A088T-N109G-G131H-T158S-N243V-S248N-K256R, A088T-N109G-G131H-T158S-S248N-K256R, A088T-N109G-G131H-T158S-S248N-K256R-L257G, A088T-N109G-G131H-V149A-K256R-L257G, A088T-N109G-N218S-K256R-L257G, A088T-N109G-N218S-N243V-K256R, A088T-N109G-N218S-N243V-L257G, A088T-N109G-N218S-N243V-S248N, A088T-N109G-N218S-N243V-S248N-K256R-L257G, A088T-N109G-N218S-S248N-K256R, A088T-N109G-N243V-K256R, A088T-N109G-N243V-S248N-K256R, A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S, A088T-N109G-T158S-K256R-L257G, A088T-N109G-T158S-N218S-N243V-K256R, A088T-N109G-T158S-N218S-N243V-K256R-L257G, A088T-N109G-T158S-N218S-N243V-S248N-K256R, A088T-N109G-T158S-N218S-N243V-S248N-L257G, A088T-N109G-T158S-N218S-S248N, A088T-N109G-T158S-S248N, A088T-N109G-T158S-S248N, A088T-N218S-N243V-K256R, A088T-N218S-N243V-L257G, A088T-N218S-N243V-S248N, A088T-N218S-N243V-S248N-K256R, A088T-N218S-N243V-S248N-K256R, A088T-N218S-S248N, A088T-N218S-S248N-L257G, A088T-S248N-K256R-L257G, A088T-T158S-K256R, A088T-T158S-N218S-N243V-K256R, A088T-T158S-N218S-N243V-L257G, A088T-T158S-N218S-N243V-S248N, A088T-T158S-N218S-N243V-S248N-K256R, A088T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-T158S-N218S-N243V-S248N-L257G, A088T-T158S-N218S-S248N, A088T-T158S-N218S-S248N-K256R, A088T-T158S-N218S-S248N-L257G, A088T-T158S-N218S-S248N-L257G-Q275K, A088T-T158S-N243V, A088T-T158S-N243V-K256R, A088T-T158S-N243V-S248N, A088T-T158S-S248N-K256R-L257G, A088T-T158S-S248N-L257G, A088T-V147I-N218S-N243V-K256R-L257G, A116T-G131H-L257G, A116T-G131H-N218S-N243V, A116T-G131H-N218S-N243V-K256R-L257G, A116T-G131H-N218S-N243V-S248N-K256R-L257G, A116T-G131H-N218S-S248N, A116T-G131H-N218S-S248N-K256R, A116T-G131H-N243V-S248N-K256R, A116T-G131H-T158S-N218S-N243V-S248N, A116T-G131H-T158S-N218S-S248N, A116T-G131H-T158S-N243V-K256R, A116T-G131H-V130I-N218S-N243V-S248N, A116T-K141E-N218S-N243V-S248N-K256R-L257G, A116T-K256R, A116T-N218S-N243V-S248N, A116T-N218T-N243V-S248N, A116T-N243V-K256R-L257G, A116T-S248N-K256R, A116T-T158S-N218S-K256R, A116T-T158S-N218S-K256R-L257G, A116T-T158S-N218S-N243V-L257G, A116T-T158S-N218S-N243V-S248N, A116T-T158S-N218S-S248N-L257G, G024S-G053S-N078S-G097A-N101S, G053S-A088T-N109G-A116T-G131H-T158S-G169S-N218S-S248N-K256R-L257G, G131H-K141R-T158S-N218S-K256R, G131H-K256R, G131H-N218S-K256R-L257G, G131H-N218S-N243V-S248N-L257G, G131H-N218S-S248N-K256R, G131H-N243V-S248N, G131H-N243V-S248N-L257G, G131H-T158S-N218S, G131H-T158S-N218S-N240H-N243V-S248N-K256R-L257G, G131H-T158S-N218S-N243V, G131H-T158S-N218S-S248N-K256R-L257G-N269S, G131H-T158S-N243V-L257G, G131H-T158S-N243V-S248N, G131H-T158S-S248N, K256R, K256R-L257G, N109G-A116T-G131H-N218S-N243V, N109G-A116T-G131H-N218S-N243V-L257G, N109G-A116T-G131H-N218S-S248N-L257G, N109G-A116T-G131H-N218S-W241R-N243V-K256R, N109G-A116T-G131H-S248N-L257G, N109G-A116T-G131H-T158S-N218S-K256R, N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G, N109G-A116T-G131H-T158S-N218S-N243V-S248N, N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G, N109G-A116T-G131H-T158S-N243V-K256R-L257G, N109G-A116T-G131H-T158S-N243V-S248N-K256R, N109G-A116T-G131H-T158S-S248N-K256R-L257G, N109G-A116T-I234T-N243V-S248N-K256R-L257G, N109G-A116T-N218S-N243V-S248N, N109G-A116T-N243V-K256R-L257G, N109G-A116T-T158S-N218S-K237R-N243V-S248N, N109G-G131H-N218S-N243V-S248N, N109G-G131H-S248N, N109G-G131H-T158S, N109G-G131H-T158S-L257G, N109G-G131H-T158S-N218S-N243V-S248N-K256R, N109G-G131H-T158S-N218S-S248N-K256R, N109G-G131H-T158S-N218S-S248N-L257G, N109G-G131H-T158S-N243V-S248N-K256R-L257G, N109G-G131H-T158S-S248N-K256R, N109G-N218S-K256R-L257G, N109G-N218S-N243V-S248N-K256R, N109G-N218S-S248N-L257G, N109G-S248N, N109G-T158S-N218S, N109G-T158S-N218S-N243V, N109G-T158S-N243V-L257G, N218S-K256R, N218S-N243V-K256R, N218S-N243V-S248N, N218S-S248N, N218S-S248N-K256R, N243V-L257G, S003P-N109G-A116T-G131H-N218S-N243V-S248N, S003P-N109G-A116T-G131H-T158S-N218S-K256R, S105H-W106G-I107L-I108S-N109A-G110A-I111S-E112N-W113G-A114P, S248N-L257G, T158S, T158S-K256R, T158S-N218S, T158S-N218S-K256R, T158S-N218S-L233S-S248N, T158S-N218S-L257G, T158S-N218S-N243V-K256R, T158S-N218S-N243V-S248N, T158S-N218S-N243V-S248N-L257G, T158S-N218S-S248N-K256R-L257G, T158S-N218S-S248N-L257G, T158S-N243V-S248N-L257G, T158S-S248N, and V004L-A088T-G131H-T158S-N218S-S248N-L257G, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A015S-A088T-N109G-G131H-T158S-N218S-S248N, A088T-A098S-G131H-S248N-K256R-L257G, A088T-A116T-G131H-N218S-N243V-K256R-L257G, A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S-L257G, A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-A116T-N218S-L257G, A088T-A116T-T158S-K256R, A088T-A116T-T158S-S248N-K256R-L257G, A088T-G131H-K141E-N218S-N243V-S248N-L257G, A088T-G131H-K256R, A088T-G131H-N218S-K256R, A088T-G131H-N218S-N243V-S248N-K256R, A088T-G131H-N218S-N243V-S248N-L257G, A088T-G131H-N218S-S248N-K256R, A088T-G131H-N218S-S248N-K256R, A088T-G131H-T158S-S248N-K256R, A088T-G131H-T158S-S248N-K256R-L257G, A088T-I107T-N109G-G131H-N218S-S248N-K256R, A088T-N109G-A116T-G131H-D140G-T158S-N218S-N243V-K256R, A088T-N109G-A116T-G131H-N218S-N243V-K256R, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G, A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-I268V, A088T-N109G-A116T-G131H-V149A-N218S-S248N-K256R-L257G, A088T-N109G-A116T-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-T158S-K256R-L257G, A088T-N109G-A116T-T158S-N218S-N243V-L257G, A088T-N109G-D140G-N243V, A088T-N109G-G131H-D140G-T158S-N243V-S248N-K256R, A088T-N109G-G131H-K141E-T158S-N218S-K256R, A088T-N109G-G131H-N218S-S248N, A088T-N109G-G131H-N218S-S248N-K256R-Q271R, A088T-N109G-G131H-N218S-S248N-L257G, A088T-N109G-G131H-T158S-K256R, A088T-N109G-G131H-T158S-N218S-S248N-K256R, A088T-N109G-G131H-V149L-T158S-K256R-L257G, A088T-N109G-T158S-N218S, A088T-N109G-T158S-N218S-K256R-L257G-Q271K, A088T-N109G-T158S-N218S-L257G, A088T-N109G-T158S-S248N-K256R, A088T-N218S-S248N-L257G-Q271R, A088T-T158S-N218S-K256R-L257G, A088T-T158S-N218S-N243V-K256R, A088T-Y104H-A116T-G131H-N218S-N243V, A116T-G131H-K141E-N218S-N243V-S248N-L257G, A116T-G131H-N218S-N243V-S248N-K256R, A116T-G131H-T158S-N218S-S248N-L257G-N269D, A116T-G131H-T158S-N218S-S248N-Q271R, A116T-G131H-T158S-N243V-S248N, A116T-G157E-T158S-N243V-S248N-K256R, A116T-T158S-N218S, G131H-N218S-L257G, G131H-N218S-S248N, G131H-T158S-N218S-N243V-S248N-K256R-L257G, G131H-T158S-N218S-N243V-S248N-L257G, G131H-T158S-N218S-S248N-I268V, I107T-N109G-G131H-N218S-L257G, L090I-N109G-T158S-N243V, L257G, N109G-A116T-G131H-T158S-N218S-K256R-L257G-Q271R, N109G-A116T-N218S-W241R-N243V-S248N-K256R-L257G, N109G-G131H-K141E-L257G, N109G-G131H-N218S-N243V, N109G-T158S-N218S-N243V-L257G, N109G-T158S-N218S-S248N-K256R, N109G-T158S-N243V-S248N-K256R-L257G, N218S-S248N-K256R-L257G, S003P-N109G-G131H-T158S-L257G, S003P-S248N-L257G, T158S-S248N-K256R-L257G, V004A-A088T-G131H-N218S-N243V-S248N-L257G, Y006H-N218S-N243V-S248N, Y104H-N109G-G131H-N243V-S248N, A088T-A116T-T158S-N218S-N243V-S248N-K256R, A088T-A116T-T158S-N243V, A088T-G131H-T158S-N218S-I234T-S248N-L257G, A088T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-G131H-V149L-T158S-N243V-S248N-K256R-L257G, A088T-I107T-N109G-G131H-N218S-A223G-S248N-K256R, A088T-K213N-N243V-S248N-K256R, A088T-K256R-L257G, A088T-N109G-A116T-G131H-A232S-N243V-K256R, A088T-N109G-A116T-G131H-D140G-S248N-L257G, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N, A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G, A088T-N109G-A116T-M124I-G131H-T158S-N218S-S248N-L257G, A088T-N109G-A116T-V148A-N218S-N243V, A088T-N109G-G131H-N218S-N243V-S248N, A088T-N109G-N218S-S248N-T255K-K256R-L257G, A088T-T158S-N218S-L257G, A088T-T158S-N218S-Q245K-S248N-K256R, A088T-T158S-N218S-S248N-K256R, A116T-G131H-N218S-N243V-K256R, A116T-G131H-N218S-W241R-N243V-S248N-K256R-L257G, A116T-G131H-T158S-N218S-L257G, A116T-G131H-V150A-T158S-N243V-S248N-K256R-L257G, I107T-G131H-T158S-N243V-S248N-K256R-L257G, N109G-A116T-K141E-T158S-N218S-N243V-L257G, N109G-A116T-T158S-N218S-N243V-S248N, T158S-N243V-S248N-K256R, T158S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-G146C, A088T-A116T-N218S, A088T-A116T-T158S-N243V-K256R-L257G, A088T-A138E-N218S-N243V-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243F-S248N, A088T-T158S-V203I-N218S-K256R-L257G, A116T-D140G-T158S-N218S-N243V-S248N, A088T-A116T-T158S-K256R-L257G, A088T-A116T-T158S-N218S-N243V-S248N-E251K-K256R-L257G, A088T-I108T-N109G-G131H-T158S-N218S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-K141E-N218S, A088T-N109G-W241R-S248N-K256R, and G065D-A088T-G131H-N243V-S248N, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-N109G-A116T-T158S-N243V-L257G, A116T-N218S-N243V-L257G-N269S, A088T-A116T-K256R, A088T-G131H-K256R, A088T-N109G-A116T-T158S-S248N-K256R-L257G, A088T-N109G-T158S-L257G, A088T-A116T-G131H-T158S-N218S-N243V-K256R-A273T, A088T-A116T-N243V-L257G, A088T-A116T-S248N-K256R-L257G, A088T-A116T-T158S-N243V-L257G, A088T-A116T-T158S-N243V-S248N-L257G, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-N243V-L257G, A088T-N109G-A116T-G131H-T158S-L257G, A088T-N109G-A116T-T158S-N218S-L257G, A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-N218S-K256R-L257G, A088T-N109G-N218S-S248N-L257G, A088T-T158S-N218S-N243V-K256R-I268V, A088T-T158S-N218S-S248N-L257G, A116T-N218S-K256R-L257G, N109G-A116T, N109G-A116T-G131H-T158S-L257G, N109G-A116T-N243V, N109G-A116T-N243V-K256R, N109G-A116T-T158S-L257G, N109G-K256R, N109G-N243V-K256R-L257G, S003P-N109G-G131H-T158S-K256R, A088T-A116T, A088T-A116T-G131H-N218S-K256R-L257G, A088T-A116T-G131H-N218S-L257G, A088T-A116T-G131H-N218S-N243V-S248N-L257G, A088T-A116T-G131H-N243V-K256R-L257G, A088T-A116T-G131H-N243V-S248N-K256R-L257G, A088T-A116T-G131H-T158S-N218S-N243V, A088T-A116T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-A116T-G131H-T158S-N218S-N243V-S248N, A088T-A116T-G131H-T158S-S248N-K256R-L257G, A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-N218S-N243V-L257G, A088T-A116T-N218S-N243V-S248N-K256R-L257G, A088T-A116T-N218S-N243V-S248N-K256R-Q275R, A088T-A116T-T158S-A216S-N218S-N243V-K256R-L257G, A088T-A116T-T158S-K256R, A088T-A116T-T158S-N218S-L257G, A088T-A116T-T158S-N218S-N243V, A088T-A116T-T158S-N218S-N243V-K256R, A088T-A116T-T158S-N218S-N243V-K256R-L257G, A088T-A116T-T158S-N218S-N243V-K256R-N269S, A088T-A116T-T158S-N243V, A088T-A116T-T158S-N243V-K256R, A088T-A116T-V147I-T158S-N218S-N243V-L257G, A088T-G131H-K256R-L257G, A088T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-G131H-S248N-K256R-L257G, A088T-G131H-T158S-N218S-L257G, A088T-G131H-T158S-N218S-N243V-L257G, A088T-I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N, A088T-I107T-N109G-G131H-N218S-S248N-K256R, A088T-N109G-A116T-G131H-A153S-N218S-S248N-L257G, A088T-N109G-A116T-G131H-K256R-L257G, A088T-N109G-A116T-G131H-N218S-K256R-L257G, A088T-N109G-A116T-G131H-N218S-L257G, A088T-N109G-A116T-G131H-N218S-N243V-K256R, A088T-N109G-A116T-G131H-N218S-N243V-L257G, A088T-N109G-A116T-G131H-N243V-L257G, A088T-N109G-A116T-G131H-S248N-L257G, A088T-N109G-A116T-G131H-T158S-N218S, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-N109G-A116T-N218S-K256R-L257G, A088T-N109G-A116T-N218S-L257G, A088T-N109G-A116T-N218S-N243V, A088T-N109G-A116T-N218S-N243V-L257G, A088T-N109G-A116T-N218T-K256R, A088T-N109G-A116T-N218T-K256R-L257G, A088T-N109G-A116T-N243V, A088T-N109G-A116T-N243V-K256R-L257G, A088T-N109G-A116T-N243V-K256R-L257G-N269D, A088T-N109G-A116T-T158S, A088T-N109G-A116T-T158S, A088T-N109G-A116T-T158S-L257G, A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G, A088T-N109G-A116T-T158S-N218S-N243V-K256R-L257G, A088T-N109G-A116T-T158S-N218S-N243V-S248N, A088T-N109G-A116T-T158S-N243V-S248N-L257G, A088T-N109G-G131H-A138V-T158S-N218S-N243V-S248N-L257G, A088T-N109G-G131H-L257G, A088T-N109G-G131H-N218S, A088T-N109G-G131H-N218S-N243V-L257G, A088T-N109G-G131H-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-N218S-S248N-K256R-L257G, A088T-N109G-G131H-T158S-N218S-K256R, A088T-N109G-G131H-T158S-N218S-N243V, A088T-N109G-G131H-T158S-N243V-K256R-L257G, A088T-N109G-G131H-T158S-N243V-L257G, A088T-N109G-G131H-T158S-N243V-S248N-L257G, A088T-N109G-G131H-V149A-K256R-L257G, A088T-N109G-N218S-N243V-L257G, A088T-N109G-N218S-N243V-S248N-L257G, A088T-N109G-N218S-S248N-K256R-L257G, A088T-N109G-N243V, A088T-N109G-N243V-K256R-L257G, A088T-N109G-N243V-L257G, A088T-N109G-N243V-S248N-L257G, A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S-K256R, A088T-N109G-T158S-N218S-N243V-K256R-Q275R, A088T-N109G-T158S-N243V, A088T-N109G-T158S-N243V-K256R-I268V, A088T-N109G-T158S-N243V-L257G, A088T-N109G-T158S-N243V-S248N-L257G, A088T-N218S-N243V-L257G, A088T-N218S-N243V-S248N-K256R-L257G, A088T-N218S-S248N, A088T-T158S-N218S-N243V-K256R-L257G, A088T-V143A-T158S-K256R, A088T-V147I-N218S-N243V-K256R-L257G, A114S-A116T-N218S-N243V-S248N-K256R-L257G, A116T-G131H-N218S-N243V-S248N-K256R-L257G, A1T-G131H-N218S-N243V-S248N-L257G, A116T-G131H-N243V-K256R, A116T-G131H-N243V-L257G, A116T-G131H-N243V-S248N-K256R, A116T-G131H-S248N-L257G, A116T-G131H-T158S-N218S-I234T-N243V-S248N-K256R, A116T-G131H-T158S-N218S-N243V-S248N-L257G, A116T-N218S-L257G, A116T-T158S-N218S-K256R-L257G, A116T-T158S-N218S-S248N, A116T-T158S-N218S-S248N-L257G-Q271R, A116T-T158S-N243V-S248N-L257G, A116T-T158S-S248N-L257G, G131H-N218S-S248N-K256R-L257G, I107T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, N109G-A116T-G131H-A137V-T158S-S248N-K256R-L257G, N109G-A116T-G131H-A151S-N218S-K256R-L257G, N109G-A116T-G131H-N218S-K256R-L257G, N109G-A116T-G131H-N218S-N243V-L257G, N109G-A116T-G131H-N243V-L257G, N109G-A116T-G131H-S248N-L257G, N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G, N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, N109G-A116T-K256R-L257G, N109G-A116T-N218S-N243V-K256R, N109G-A116T-N218S-N243V-S248N-K256R-L257G, N109G-A116T-N218S-N243V-S248N-L257G, N109G-A116T-N243V-S248N-K256R, N109G-A116T-S248N-L257G, N109G-A116T-T158S-G211V-N243V-S248N-K256R, N109G-A116T-T158S-N218S, N109G-A116T-T158S-N218S-N243V-L257G, N109G-A116T-T158S-N218S-N243V-S248N, N109G-G131H-N218S-K237N, N109G-G131H-N218S-K256R-L257G, N109G-G131H-N218S-L257G, N109G-G131H-N218S-N243V-L257G, N109G-G131H-N243V-K256R-L257G, N109G-G131H-N243V-S248N-L257G, N109G-G131H-S248N-K256R, N109G-G131H-T158S-K256R-L257G, N109G-G131H-T158S-N218S-K256R-L257G, N109G-G131H-T158S-N243V, N109G-N218S, N109G-N218S-N243V-L257G, N109G-T158S-N218S-K256R-L257G, N109G-T158S-N218S-L257G, N109G-T158S-N218S-N243V-K256R, N109G-T158S-N243V-L257G, N243V-K256R-L257G, N243V-L257G, S003F-A088T-N109G-A116T-T158S-N243V-K256R-L257G, T158S-N218S-L233S, T158S-N218S-N243V-S248N-K256R, V004A-N109G-A116T-T158S-N218S-S248N-L257G, Y006H-A116T-G131H-T158S-N218S-N243V-S248N-K256R-A272G, A088T-A098S-N218S-K256R, A088T-A116T-G131H-K256R-L257G-L267M, A088T-A116T-G131H-L257G, A088T-A116T-G131H-N218S-A274T, A088T-A116T-G131H-N218S-K256R, A088T-A116T-G131H-N218S-N243V-K256R, A088T-A116T-G131H-N218S-N243V-K256R-L257G, A088T-A116T-G131H-N218S-N243V-K256R-L257G, A088T-A116T-G131H-N218S-N243V-L257G, A088T-A116T-G131H-N218S-N243V-S248N-K256R, A088T-A116T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-N218S-S248N-L257G, A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-N243V-L257G, A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-N243V-S248N-A274V, A088T-A116T-G131H-N243V-S248N-K256R-L257G, A088T-A116T-G131H-S248N-K256R-L257G, A088T-A116T-G131H-S248N-L257G, A088T-A116T-G131H-T158S-K256R-L257G, A088T-A116T-G131H-T158S-K256R-L257G, A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S, A088T-A116T-G131H-T158S-N218S-K256R, A088T-A116T-G131H-T158S-N218S-K256R-L257G, A088T-A116T-G131H-T158S-N218S-K256R-L257G, A088T-A116T-G131H-T158S-N218S-L257G, A088T-A116T-G131H-T158S-N218S-N243V-K256R, A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-A116T-G131H-T158S-N218S-S248N-K256R, A088T-A116T-G131H-T158S-N218S-S248N-L257G, A088T-A116T-G131H-T158S-N243V-K256R-L257G, A088T-A116T-G131H-T158S-N243V-L257G, A088T-A116T-G131H-T158S-N243V-S248N-K256R, A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-A116T-K256R, A088T-A116T-N218S-K256R, A088T-A116T-N218S-N243V-K256R, A088T-A116T-N218S-N243V-K256R-L257G, A088T-A116T-N218S-N243V-S248N-K256R, A088T-A116T-N218S-N243V-S248N-L257G, A088T-A116T-N243V-K256R, A088T-A116T-N243V-L257G, A088T-A116T-N243V-S248N-K256R, A088T-A116T-N243V-S248N-K256R-L257G, A088T-A116T-S248N, A088T-A116T-S248N-K256R, A088T-A116T-S248N-L257G, A088T-A116T-T158S-N218S, A088T-A116T-T158S-N218S-K256R, A088T-A116T-T158S-N218S-K256R, A088T-A116T-T158S-N218S-K256R-L257G, A088T-A116T-T158S-N218S-N243V-K256R, A088T-A116T-T158S-N218S-N243V-S248N, A088T-A116T-T158S-N218S-N243V-S248N, A088T-A116T-T158S-N218S-N243V-S248N-K256R, A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-A116T-T158S-N218S-N243V-S248N-L257G, A088T-A116T-T158S-N243V-K256R-L257G, A088T-G131H, A088T-G131H-N218S-K256R-L257G, A088T-G131H-N218S-N243V-K256R, A088T-G131H-N218S-N243V-K256R, A088T-G131H-N218S-N243V-L257G, A088T-G131H-N218S-N243V-L257G, A088T-G131H-N218S-S248N, A088T-G131H-N218S-S248N-K256R, A088T-G131H-N218S-S248N-K256R-L257G, A088T-G131H-N218S-S248N-L257G, A088T-G131H-N218T-L257G, A088T-G131H-N243V-L257G, A088T-G131H-N243V-S248N-K256R, A088T-G131H-S248N, A088T-G131H-S248N-L257G, A088T-G131H-T158S-N218S-K256R, A088T-G131H-T158S-N218S-K256R-L257G, A088T-G131H-T158S-N218S-N243V-K256R, A088T-G131H-T158S-N218S-N243V-K256R, A088T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-G131H-T158S-N218S-S248N, A088T-G131H-T158S-N218S-S248N-K256R, A088T-G131H-T158S-N218S-S248N-K256R, A088T-G131H-T158S-N218S-S248N-L257G-I268V, A088T-G131H-T158S-N243V, A088T-G131H-T158S-N243V-S248N, A088T-G131H-T158S-N243V-S248N, A088T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-N109G-A116T, A088T-N109G-A116T-G131H-D140G-T158S-N218S-N243V-K256R, A088T-N109G-A116T-G131H-K256R, A088T-N109G-A116T-G131H-N218S, A088T-N109G-A116T-G131H-N218S, A088T-N109G-A116T-G131H-N218S-K256R, A088T-N109G-A116T-G131H-N218S-L257G, A088T-N109G-A116T-G131H-N218S-N243V, A088T-N109G-A116T-G131H-N218S-N243V-K256R-L257G, A088T-N109G-A116T-G131H-N218S-N243V-L257G, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-N218S-S248N-L257G, A088T-N109G-A116T-G131H-N218S-S248N-L257G, A088T-N109G-A116T-G131H-N243V-K256R-L257G, A088T-N109G-A116T-G131H-N243V-S248N, A088T-N109G-A116T-G131H-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-N243V-S248N-L257G, A088T-N109G-A116T-G131H-S248N-K256R, A088T-N109G-A116T-G131H-S248N-K256R-L257G, A088T-N109G-A116T-G131H-S248N-L257G, A088T-N109G-A116T-G131H-T158S, A088T-N109G-A116T-G131H-T158S-N218S, A088T-N109G-A116T-G131H-T158S-N218S-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218S-S248N, A088T-N109G-A116T-G131H-T158S-N218S-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G, A088T-N109G-A116T-G131H-T158S-N218S-S248N-L257G, A088T-N109G-A116T-G131H-T158S-N218T-N243V, A088T-N109G-A116T-G131H-T158S-N243V-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N243V-S248N, A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G, A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-S248N-L257G, A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S, A088T-N109G-A116T-N218S-N243V, A088T-N109G-A116T-N218S-N243V-K256R, A088T-N109G-A116T-N218S-N243V-K256R-L257G, A088T-N109G-A116T-N218S-N243V-L257G, A088T-N109G-A116T-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-N243V-S248N, A088T-N109G-A116T-N243V-S248N-K256R, A088T-N109G-A116T-N243V-S248N-K256R-L257G, A088T-N109G-A116T-N243V-S248N-K256R-L257G, A088T-N109G-A116T-N243V-S248N-L257G, A088T-N109G-A116T-S248N-K256R, A088T-N109G-A116T-S248N-K256R-L257G, A088T-N109G-A116T-T158S-K256R, A088T-N109G-A116T-T158S-N218S, A088T-N109G-A116T-T158S-N218S-K256R-L257G, A088T-N109G-A116T-T158S-N218S-L257G, A088T-N109G-A116T-T158S-N218S-N243V, A088T-N109G-A116T-T158S-N218S-N243V-L257G, A088T-N109G-A116T-T158S-N218S-N243V-S248N, A088T-N109G-A116T-T158S-N218S-N243V-S248N-K256R, A088T-N109G-A116T-T158S-N218S-N243V-S248N-L257G, A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-A116T-T158S-N218S-S248N-K256R, A088T-N109G-A116T-T158S-N218S-S248N-L257G, A088T-N109G-A116T-T158S-N243V-K256R, A088T-N109G-A116T-T158S-N243V-S248N, A088T-N109G-A116T-T158S-S248N, A088T-N109G-A116T-T158S-S248N-K256R, A088T-N109G-A116T-T158S-S248N-L257G, A088T-N109G-G131H-N218S-K256R, A088T-N109G-G131H-N218S-K256R, A088T-N109G-G131H-N218S-N243V-K256R, A088T-N109G-G131H-N218S-N243V-K256R, A088T-N109G-G131H-N218S-N243V-K256R-L257G, A088T-N109G-G131H-N218S-N243V-S248N-K256R, A088T-N109G-G131H-N218S-S248N, A088T-N109G-G131H-N218S-S248N-L257G, A088T-N109G-G131H-N243V-K256R-L257G, A088T-N109G-G131H-N243V-K256R-L257G, A088T-N109G-G131H-N243V-L257G, A088T-N109G-G131H-N243V-L257G, A088T-N109G-G131H-N243V-S248N-K256R, A088T-N109G-G131H-N243V-S248N-K256R-L257G, A088T-N109G-G131H-N243V-S248N-L257G, A088T-N109G-G131H-S248N-L257G, A088T-N109G-G131H-T158S-K256R-L257G, A088T-N109G-G131H-T158S-N218S-L257G, A088T-N109G-G131H-T158S-N218S-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N, A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R, A088T-N109G-G131H-T158S-N218S-S248N-K256R, A088T-N109G-G131H-T158S-N218S-S248N-L257G, A088T-N109G-G131H-T158S-N243V, A088T-N109G-G131H-T158S-N243V-S248N, A088T-N109G-G131H-T158S-N243V-S248N-K256R, A088T-N109G-G131H-T158S-N243V-S248N-K256R, A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-S248N-K256R-L257G, A088T-N109G-G131H-T158S-W241R-S248N-K256R, A088T-N109G-G131H-V148A-N218S-N243V-K256R-L257G, A088T-N109G-G154A-N155P-E156T-G157L-T158M-S159E-G160E-S161L, A088T-N109G-N218S-K256R, A088T-N109G-N218S-N243V-L257G, A088T-N109G-N218S-N243V-S248N, A088T-N109G-N243V-K256R, A088T-N109G-N243V-S248N, A088T-N109G-N243V-S248N-K256R-L257G, A088T-N109G-S248N, A088T-N109G-T158S-N218S, A088T-N109G-T158S-N218S-K256R, A088T-N109G-T158S-N218S-K256R-L257G, A088T-N109G-T158S-N218S-L257G, A088T-N109G-T158S-N218S-N243V-S248N-K256R, A088T-N109G-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-T158S-N218S-S248N-K256R, A088T-N109G-T158S-N218S-S248N-K256R-L257G, A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-K256R, A088T-N109G-T158S-N243V-S248N-L257G, A088T-N109G-T158S-S248N-K256R-L257G, A088T-N109G-T158S-S248N-L257G, A088T-N109G-T158S-S248N-L257G, A088T-N109G-V147A-N218S-N243V-K256R, A088T-N218S-L257G-I268V, A088T-N218S-N243V, A088T-N218S-N243V-S248N, A088T-N218S-N243V-S248N-N269S, A088T-N218S-S248N-K256R, A088T-N218S-S248N-L257G-Q271R, A088T-N243V, A088T-N243V, A088T-N243V-K256R, A088T-N243V-S248N-K256R, A088T-N243V-S248N-K256R-L257G, A088T-S248N, A088T-T158S-N218S-K256R-L257G, A088T-T158S-N218S-N243V-K256R, A088T-T158S-N218S-N243V-L257G, A088T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-T158S-N243V-K256R-L257G, A088T-T158S-N243V-K256R-L257G-Q271H, A088T-T158S-N243V-S248N, A088T-T158S-N243V-S248N-L257G, A088T-T158S-S248N, A088T-V147A-K256R, A116T-G131H-K256R, A116T-G131H-N218S, A116T-G131H-N218S-K256R-L257G, A116T-G131H-N218S-L257G, A116T-G131H-N218S-N243V, A116T-G131H-N218S-S248N-K256R, A116T-G131H-N218S-S248N-K256R-L257G, A116T-G131H-N243V-S248N, A116T-G131H-N243V-S248N-L257G, A116T-G131H-S248N-K256R, A116T-G131H-T158S-A231V-N243V-L257G, A116T-G131H-T158S-N218S-K256R, A116T-G131H-T158S-N218S-K256R-L257G, A116T-G131H-T158S-N218S-N243V-K256R-L257G, A116T-G131H-T158S-N218S-N243V-S248N-K256R, A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A116T-G131H-T158S-N218S-S248N, A116T-G131H-T158S-N243V-L257G, A116T-G131H-T158S-N243V-S248N-K256R, A116T-G131H-T158S-S248N-K256R, A116T-G131H-T158S-S248N-L257G, A116T-G131H-V143F-T158S-N218S, A116T-K256R, A116T-N218S, A116T-N218S-K256R, A116T-N218S-N243V-L257G, A116T-N218S-N243V-S248N-K256R, A116T-N218S-N243V-S248N-K256R-L257G, A116T-N218S-N243V-S248N-L257G, A116T-N218S-S248N-L257G, A116T-N243V, A116T-N243V-K256R-L257G, A116T-N243V-S248N, A116T-N243V-S248N-K256R-L257G, A116T-S248N-K256R-L257G, A116T-T158S-K256R-L257G, A116T-T158S-N218S, A116T-T158S-N218S-K256R, A116T-T158S-N218S-N243V, A116T-T158S-N218S-N243V-S248N, A116T-T158S-N218S-S248N-K256R, A116T-T158S-N218S-S248N-K256R-L257G, A116T-T158S-N243V-K256R, A116T-T158S-N243V-L257G, A116T-T158S-N243V-S248N, A116T-T158S-S248N-K256R-L257G, G131H-K141R-T158S-N218S-K256R, G131H-N218S, G131H-N218S-K256R, G131H-N218S-N243V-K256R-L257G, G131H-N218S-N243V-S248N, G131H-N218S-N243V-S248N-L257G, G131H-N243V-S248N-K256R, G131H-N243V-S248N-K256R-L257G, G131H-S248N, G131H-T158S-I234T-N243V-K256R, G131H-T158S-N218S-K256R-L257G, G131H-T158S-N218S-N243V, G131H-T158S-N218S-N243V-S248N-L257G, G131H-T158S-N218S-S248N-K256R-L257G, G131H-T158S-N218S-S248N-L257G, G131H-T158S-N243V-K256R, G131H-T158S-N243V-S248N-L257G, N109G, N109G-A116T-G131H-A144V-T158S-S248N-K256R-L257G, N109G-A116T-G131H-K256R-L257G, N109G-A116T-G131H-N218S-N243V-K256R, N109G-A116T-G131H-N218S-N243V-K256R-L257G, N109G-A116T-G131H-N218S-S248N-K256R, N109G-A116T-G131H-N218S-S248N-L257G, N109G-A116T-G131H-N243V-K256R, N109G-A116T-G131H-N243V-S248N, N109G-A116T-G131H-N243V-S248N-K256R-L257G, N109G-A116T-G131H-S248N, N109G-A116T-G131H-S248N-K256R, N109G-A116T-G131H-T158S-K256R, N109G-A116T-G131H-T158S-K256R-L257G, N109G-A116T-G131H-T158S-N218S-K256R, N109G-A116T-G131H-T158S-N218S-K256R-L257G, N109G-A116T-G131H-T158S-N218S-L257G, N109G-A116T-G131H-T158S-N218S-N243V-K256R, N109G-A116T-G131H-T158S-N218S-N243V-S248N-L257G, N109G-A116T-G131H-T158S-N218S-S248N, N109G-A116T-G131H-T158S-N243V-L257G, N109G-A116T-G131H-T158S-N243V-S248N, N109G-A116T-G131H-T158S-S248N, N109G-A116T-G131H-T158S-S248N-K256R, N109G-A116T-G131H-T158S-S248N-K256R-L257G, N109G-A116T-G131H-V149A-T158S-N218S-N243V-S248N-L257G, N109G-A116T-N218S, N109G-A116T-N218S-K256R, N109G-A116T-N218S-N243V-K256R-L257G, N109G-A116T-N218S-N243V-S248N-I268V, N109G-A116T-N243V-K256R-L257G, N109G-A116T-N243V-S248N, N109G-A116T-N243V-S248N-L257G, N109G-A116T-T158S, N109G-A116T-T158S-N218S-N243V-K256R-L257G, N109G-A116T-T158S-N218S-N243V-S248N-K256R-L257G, N109G-A116T-T158S-N218S-N243V-S248N-L257G, N109G-A116T-T158S-N218S-S248N-K256R-L257G, N109G-A116T-T158S-N243V-K256R-L257G, N109G-A116T-T158S-N243V-L257G, N109G-A116T-T158S-N243V-S248N-L257G, N109G-A116T-T158S-Q275R, N109G-A116T-T158S-S248N-K256R-L257G, N109G-G131H-L257G, N109G-G131H-N218S-N243V-S248N-K256R-L257G, N109G-G131H-N218S-N243V-S248N-L257G, N109G-G131H-N218S-S248N-K256R-L257G, N109G-G131H-N243V-K256R, N109G-G131H-S145F-N218S-N243V-K256R-L257G, N109G-G131H-S248N-K256R-L257G, N109G-G131H-S248N-L257G, N109G-G131H-T158S-K256R, N109G-G131H-T158S-N218S-L257G, N109G-G131H-T158S-N218S-N243V, N109G-G131H-T158S-N218S-N243V-K256R, N109G-G131H-T158S-N218S-N243V-K256R-L257G, N109G-G131H-T158S-N218S-N243V-S248N-L257G, N109G-G131H-T158S-N218S-S248N-K256R, N109G-G131H-T158S-N218S-S248N-K256R-L257G-A274T, N109G-G131H-T158S-N218S-S248N-L257G, N109G-G131H-T158S-N243V-S248N, N109G-G131H-T158S-N243V-S248N-K256R-L257G, N109G-G131H-T158S-S248N-K256R-L257G, N109G-G131H-T158S-S248N-L257G, N109G-N218S-K256R-L257G, N109G-N218S-L257G, N109G-N218S-N243V-K256R, N109G-N218S-N243V-S248N-S260F, N109G-N218S-S248N, N109G-N243V-K256R, N109G-N243V-L257G, N109G-N243V-S248N, N109G-N243V-S248N-K256R-L257G, N109G-S182F-S204F-S207L-N218S-S236F-S248N-L257G, N109G-S248N-K256R, N109G-T158S-K256R, N109G-T158S-N218S-N243V-K256R-L257G, N109G-T158S-N218S-S248N-L257G, N109G-T158S-N243V, N109G-T158S-N243V-K256R, N109G-T158S-N243V-S248N, N109G-T158S-N243V-S248N-K256R, N109G-T158S-S248N-K256R, N109G-T158S-S248N-L257G, N218S, N218S-N243V-S248N-K256R, N218S-S248N-L257G, N243V-K256R, N243V-S248N-K256R, N243V-S248N-K256R-L257G, N243V-S248N-L257G-Q271R, S003P-A116T-T158S-S248N-K256R, S248N, T158S-N218S, T158S-N218S-A272V, T158S-N218S-L257G, T158S-N218S-N243V-K256R-L257G, T158S-N218S-N243V-L257G, T158S-N218S-S248N-K256R-L257G, T158S-N243V-K256R, T158S-N243V-K256R-L257G, T158S-N243V-S248N-K256R, V004A-N109G-A116T-G131H-S248N-K256R-L257G, and Y006H-A116T-G131H-S248N, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A015S-A088T-N109G-G131H-T158S-N218S-S248N, A088T, A088T-A116T-G131H-N218S-N243V-S248N, A088T-A116T-G131H-N218S-S248N, A088T-A116T-G131H-N218S-S248N, A088T-A116T-G131H-N218S-S248N-K256R-L257G, A088T-A116T-G131H-N243V, A088T-A116T-G131H-N243V-K256R, A088T-A116T-G131H-N243V-S248N, A088T-A116T-G131H-S248N-K256R, A088T-A116T-G131H-T158S-N218S-N243V-S248N, A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-A116T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-A116T-G131H-T158S-N218S-S248N, A088T-A116T-G131H-T158S-N218S-S248N-K256R, A088T-A116T-G131H-T158S-N243V, A088T-A116T-G131H-T158S-N243V-K256R, A088T-A116T-G131H-T158S-N243V-S248N, A088T-A116T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-A116T-G131H-T158S-S248N, A088T-A116T-G131H-T158S-S248N-K256R, A088T-A116T-G131H-T158S-S248N-L257G, A088T-A116T-G131H-V147A-T158S-N218S-N243V-S248N-L257G, A088T-A116T-N218S-I268V, A088T-A116T-N218S-L257G, A088T-A116T-N218S-N243V-N269D, A088T-A116T-N218S-S248N, A088T-A116T-N218S-S248N, A088T-A116T-N218S-S248N-L257G, A088T-A116T-N243V-K256R-L257G, A088T-A116T-N243V-S248N-K256R, A088T-A116T-S248N-K256R, A088T-A116T-T158S-N218S, A088T-A116T-T158S-N218S-N243V-S248N-K256R-L257G, A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N218S-S248N-K256R-L257G, A088T-A116T-T158S-N243V-S248N, A088T-A116T-T158S-N243V-S248N-K256R, A088T-A116T-T158S-N243V-S248N-K256R-L257G, A088T-A116T-T158S-N243V-S248N-L257G, A088T-A116T-T158S-S248N, A088T-A116T-T158S-S248N-K256R-L257G, A088T-A116T-T158S-S248N-L257G, A088T-A116T-V143A-N218S-S248N-K256R, A088T-G131H-A138V-N218S-L257G, A088T-G131H-K141E-N218S-N243V-S248N-L257G, A088T-G131H-K256R, A088T-G131H-N218S-K256R, A088T-G131H-N218S-N243V-K256R-L257G, A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-N243V-S248N, A088T-G131H-N218S-N243V-S248N-K256R, A088T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-G131H-N218S-S248N, A088T-G131H-N218S-S248N-K256R, A088T-G131H-N243V-K256R-L257G, A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N-K256R-L257G, A088T-G131H-S248N, A088T-G131H-S248N-K256R, A088T-G131H-T158S-K256R, A088T-G131H-T158S-L257G, A088T-G131H-T158S-N218S, A088T-G131H-T158S-N218S-N243V-S248N, A088T-G131H-T158S-N218S-N243V-S248N-K256R, A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-G131H-T158S-N218S-N243V-S248N-L257G, A088T-G131H-T158S-N218S-S248N, A088T-G131H-T158S-N218S-S248N-K256R-L257G, A088T-G131H-T158S-N243V, A088T-G131H-T158S-N243V-K256R, A088T-G131H-T158S-N243V-S248N-K256R, A088T-G131H-T158S-N243V-S248N-L257G, A088T-G131H-T158S-S248N-K256R-L257G, A088T-G131H-T158S-S248N-L257G, A088T-I107T-N109G-G131H-N218S-A223G-S248N-K256R, A088T-L257G, A088T-L257G, A088T-N109G-A116T-G131H-A232S-N243V-K256R, A088T-N109G-A116T-G131H-K256R-L257G, A088T-N109G-A116T-G131H-L257G, A088T-N109G-A116T-G131H-N218S-K256R, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R, A088T-N109G-A116T-G131H-N218S-N243V-S248N-N269D, A088T-N109G-A116T-G131H-N243V-K256R, A088T-N109G-A116T-G131H-S248N, A088T-N109G-A116T-G131H-T158S-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243F-S248N, A088T-N109G-A116T-G131H-T158S-N218S-N243V, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N218T-K256R, A088T-N109G-A116T-G131H-T158S-N243V, A088T-N109G-A116T-G131H-T158S-N243V-K256R, A088T-N109G-A116T-G131H-T158S-N243V-S248N, A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-I268V, A088T-N109G-A116T-G131H-T158S-N243V-S248N-L257G, A088T-N109G-A116T-G131H-T158S-S248N, A088T-N109G-A116T-G131H-T158S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-V149A-N218S-S248N-K256R-L257G, A088T-N109G-A116T-K256R, A088T-N109G-A116T-N218S-K256R, A088T-N109G-A116T-N218S-N243V-S248N-K256R, A088T-N109G-A116T-N218S-N243V-S248N-L257G, A088T-N109G-A116T-N218S-S248N, A088T-N109G-A116T-N218S-S248N-K256R, A088T-N109G-A116T-S248N, A088T-N109G-A116T-T158S-L257G, A088T-N109G-A116T-T158S-N218S, A088T-N109G-A116T-T158S-N243V, A088T-N109G-A116T-T158S-N243V-K256R, A088T-N109G-A116T-T158S-N243V-S248N-K256R, A088T-N109G-G131H-N218S-N243V-S248N, A088T-N109G-G131H-N218S-S248N-K256R, A088T-N109G-G131H-N218S-S248N-K256R-L257G, A088T-N109G-G131H-N218S-S248N-K256R-L257G-Q275R, A088T-N109G-G131H-N218S-S248N-K256R-Q271R, A088T-N109G-G131H-N243V, A088T-N109G-G131H-N243V-K256R, A088T-N109G-G131H-N243V-S248N-K256R, A088T-N109G-G131H-S248N-K256R, A088T-N109G-G131H-S248N-L257G, A088T-N109G-G131H-T158S, A088T-N109G-G131H-T158S, A088T-N109G-G131H-T158S-K256R, A088T-N109G-G131H-T158S-K256R, A088T-N109G-G131H-T158S-N218S-N243V-K256R, A088T-N109G-G131H-T158S-N218S-N243V-K256R, A088T-N109G-G131H-T158S-N218S-N243V-K256R-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N-L257G, A088T-N109G-G131H-T158S-N243V-K256R, A088T-N109G-G131H-T158S-N243V-K256R-L257G, A088T-N109G-G131H-T158S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-S248N, A088T-N109G-G131H-T158S-S248N-L257G, A088T-N109G-G131H-V149A-K256R-L257G, A088T-N109G-G131H-V149L-T158S-K256R-L257G, A088T-N109G-K256R, A088T-N109G-K256R-L257G, A088T-N109G-K256R-L257G, A088T-N109G-L257G, A088T-N109G-N218S-K256R, A088T-N109G-N218S-N243V-K256R, A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-N243V-S248N-K256R, A088T-N109G-N218S-S248N, A088T-N109G-N218S-S248N-L257G, A088T-N109G-N243V-K256R, A088T-N109G-N243V-S248N-L257G-I268V, A088T-N109G-S248N, A088T-N109G-S248N-K256R, A088T-N109G-T158S-K256R-L257G, A088T-N109G-T158S-N218S, A088T-N109G-T158S-N218S-K256R-Q271H, A088T-N109G-T158S-N218S-N243V-K256R, A088T-N109G-T158S-N218S-N243V-K256R-L257G, A088T-N109G-T158S-N218S-N243V-L257G, A088T-N109G-T158S-N218S-N243V-S248N-K256R, A088T-N109G-T158S-N218S-S248N-N269D, A088T-N109G-T158S-N243V-K256R-L257G, A088T-N109G-T158S-N243V-S248N-A274D, A088T-N109G-T158S-N243V-S248N-K256R-L257G-N269D, A088T-N218S-K256R, A088T-N218S-N243V, A088T-N218S-N243V-K256R, A088T-N218S-N243V-K256R-L257G, A088T-N218S-N243V-S248N-K256R, A088T-N218S-N243V-S248N-L257G, A088T-N218S-S248N-L257G, A088T-N243V-S248N, A088T-N243V-S248N-K256R, A088T-N243V-S248N-L257G, A088T-S145T-T158S-S248N, A088T-S248N-K256R-L257G, A088T-S248N-L257G, A088T-T158S, A088T-T158S, A088T-T158S-K256R, A088T-T158S-L257G, A088T-T158S-N218S, A088T-T158S-N218S-K256R, A088T-T158S-N218S-N243V-K256R, A088T-T158S-N218S-N243V-K256R-L257G, A088T-T158S-N218S-N243V-L257G, A088T-T158S-N218S-S248N, A088T-T158S-N218S-S248N, A088T-T158S-N243V, A088T-T158S-N243V-K256R, A088T-T158S-N243V-S248N-L257G, A116T, A116T-G131H-L257G, A116T-G131H-N218S-K256R, A116T-G131H-N218S-N243V-K256R, A116T-G131H-N218S-N243V-K256R-L257G, A116T-G131H-N218S-N243V-S248N, A116T-G131H-N218S-N243V-S248N-K256R, A116T-G131H-N218S-S248N, A116T-G131H-T158S-K256R, A116T-G131H-T158S-N218S-L257G, A116T-G131H-T158S-N218S-N243V-K256R, A116T-G131H-T158S-N218S-N243V-L257G, A116T-G131H-T158S-N218S-N243V-S248N, A116T-G131H-T158S-N218S-S248N-L257G-N269D, A116T-G131H-T158S-N218S-S248N-Q271R, A116T-G131H-T158S-N218T-L257G, A116T-G131H-T158S-N243V-K256R, A116T-G131H-V150A-T158S-N243V-S248N-K256R-L257G, A116T-K141E-N218S-N243V-S248N-K256R-L257G, A116T-L257G, A116T-N218S-N243V-S248N, A116T-N218T-N243V-S248N, A116T-N243V-S248N-L257G, A116T-S248N, A116T-S248N-L257G, A116T-T158S-K256R, A116T-T158S-N218S-L257G, A116T-T158S-N218S-N243V-K256R, A116T-T158S-N218S-N243V-L257G, A116T-T158S-N243V, A116T-T158S-N243V-K256R-L257G, A116T-T158S-N243V-S248N-K256R, A116T-T158S-N243V-S248N-K256R-L257G, A116T-V149I-T158S-N243V-S248N-K256R-Q271H, G024S-G053S-N078S-G097A-N101S, G024S-G053S-N078S-G097A-N101S-A128S, G131H-K256R, G131H-N218S-K256R-L257G, G131H-N218S-N243V-L257G, G131H-N218S-N243V-S248N-K256R, G131H-N218S-S248N-K256R, G131H-N218S-S248N-L257G, G131H-N243V, G131H-N243V-S248N, G131H-N243V-S248N-L257G, G131H-T158S-K256R-L257G, G131H-T158S-N218S, G131H-T158S-N218S-K256R, G131H-T158S-N218S-N240H-N243V-S248N-K256R-L257G, G131H-T158S-N218S-N243V-K256R, G131H-T158S-N218S-N243V-K256R-L257G, G131H-T158S-N218S-N243V-S248N, G131H-T158S-N218S-N243V-S248N-K256R-L257G, G131H-T158S-N218S-S248N-I268V, G131H-T158S-N218S-S248N-K256R-L257G-N269S, G131H-T158S-N243V-K256R-L257G, G131H-T158S-N243V-S248N, G131H-T158S-N243V-S248N-K256R, G131H-T158S-S248N, G131H-T158S-S248N-L257G, G131H-W241L-N243V-S248N-K256R, I107T-G131H-T158S-N243V-S248N-K256R-L257G, I107T-N109G-G131H-N218S-L257G, N109G-A116T-G131H-L257G, N109G-A116T-G131H-N218S-L257G, N109G-A116T-G131H-N218S-N243V, N109G-A116T-G131H-N218S-N243V-S248N-K256R, N109G-A116T-G131H-N218S-W241R-N243V-K256R, N109G-A116T-G131H-N243V-S248N-K256R, N109G-A116T-G131H-S248N-I268V, N109G-A116T-G131H-T158S-N218S, N109G-A116T-G131H-T158S-N218S-S248N-K256R, N109G-A116T-G131H-T158S-N218S-S248N-K256R-L257G, N109G-A116T-G131H-T158S-N218S-S248N-L257G, N109G-A116T-G131H-T158S-N243V-K256R-L257G, N109G-A116T-G131H-T158S-S248N-L257G, N109G-A116T-K141E-T158S-N218S-N243V-L257G, N109G-A116T-K256R, N109G-A116T-N218S-N243V, N109G-A116T-N218S-N243V-S248N, N109G-A116T-N218S-S248N-L257G, N109G-A116T-N243V-S248N-K256R-L257G, N109G-A116T-S248N, N109G-A116T-T158S-N218S-N243V-S248N-K256R, N109G-A116T-T158S-N218S-W241R-N243V, N109G-A116T-T158S-N243V-S248N, N109G-A116T-T158S-S248N-L257G, N109G-G131H-K256R, N109G-G131H-N218S-K256R, N109G-G131H-N218S-N243V, N109G-G131H-N218S-N243V-K256R-L257G, N109G-G131H-N218S-N243V-S248N-K256R, N109G-G131H-N218S-S248N-K256R, N109G-G131H-N243V, N109G-G131H-N243V-S248N, N109G-G131H-N243V-S248N-K256R-L257G, N109G-G131H-S248N, N109G-G131H-T158S-L257G, N109G-G131H-T158S-N218S-N243V-S248N, N109G-G131H-T158S-N218S-N243V-S248N-K256R, N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G, N109G-G131H-T158S-N243V-L257G, N109G-G131H-T158S-N243V-S248N-K256R, N109G-G131H-T158S-S248N-K256R, N109G-K141E-N218S-S248N-L257G, N109G-N218S-N243V, N109G-N218S-N243V-S248N-K256R, N109G-N218S-S248N-K256R-L257G, N109G-N218S-S248N-L257G, N109G-N243V-S248N-L257G-Q275R, N109G-T158S-I268V, N109G-T158S-N218S, N109G-T158S-N218S-N243V, N109G-T158S-N218S-N243V-S248N, N109G-T158S-N218S-S248N-K256R, N109S-A116T-S248N, N218S-K256R, N218S-N243V-K256R, N218S-N243V-L257G, N218S-N243V-S248N, N218S-S248N, N218S-S248N-K256R, N218S-S248N-K256R-L257G, S003P-N109G-G131H-N218S-N243V-S248N-K256R-L257G, S248N-K256R-L257G, T158S-K256R, T158S-K256R-L257G, T158S-N218S-K256R-L257G, T158S-N218S-L233S-S248N, T158S-N243V, T158S-N243V-S248N-K256R-N269D, T158S-N243V-S248N-L257G, V004L-A116T-N218S-N243V-S248N-L257G, and Y006H-N218S-N243V-S248N, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-A098S-G131H-S248N-K256R-L257G, A088T-A116T-G131H-K256R, A088T-A116T-G131H-N218S-S248N-K256R, A088T-A116T-G131H-N243V-S248N-L257G, A088T-A116T-G131H-S248N, A088T-A116T-G131H-S248N-K256R-L257G, A088T-A116T-G131H-T158S-K256R, A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H-T158S-N218S-L257G, A088T-A116T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-A116T-K256R-L257G, A088T-A116T-K256R-L257G, A088T-A116T-N218S-N243V-Q271R, A088T-A116T-N218S-S248N-K256R, A088T-A116T-T158S, A088T-A116T-T158S, A088T-A116T-T158S-K256R, A088T-A116T-T158S-N218S-N243V-L257G, A088T-A116T-T158S-N218S-S248N-K256R, A088T-G131H, A088T-G131H-L257G, A088T-G131H-N218S-N243V-S248N-L257G, A088T-G131H-N243V-K256R, A088T-G131H-N243V-L257G, A088T-G131H-N243V-S248N, A088T-G131H-N243V-S248N-K256R, A088T-G131H-T158S-N218S-I234T-S248N-L257G, A088T-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-G131H-T158S-N243V-K256R-L257G, A088T-G131H-T158S-S248N, A088T-G131H-T158S-S248N-K256R, A088T-G131H-T158S-S248N-K256R-L257G, A088T-G131H-V149L-T158S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-K141E-N218S, A088T-N109G-A116T-G131H-N218S-N243V-S248N-Q275R, A088T-N109G-A116T-G131H-N218S-S248N, A088T-N109G-A116T-G131H-N243V-S248N-K256R, A088T-N109G-A116T-G131H-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-K256R, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N, A088T-N109G-A116T-G131H-T158S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-S248N, A088T-N109G-A116T-G131H-V149A-T158S-N218S-K256R, A088T-N109G-A116T-N218S-S248N-K256R-L257G, A088T-N109G-A116T-N243V-K256R, A088T-N109G-A116T-N243V-S248N-L257G, A088T-N109G-A116T-S248N-L257G, A088T-N109G-A116T-T158S-N212D-N243V-K256R-L257G, A088T-N109G-A137E-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-D140G-N243V, A088T-N109G-G131H-A152S-T158S-N218S-S248N-K256R, A088T-N109G-G131H-D140G-T158S-N243V-S248N-K256R, A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S, A088T-N109G-G131H-N218S-N243V, A088T-N109G-G131H-T158S-L257G, A088T-N109G-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A088T-N109G-G131H-T158S-N218S-W241R-S248N-L257G, A088T-N109G-G131H-T158S-S248N-K256R, A088T-N109G-N218S-S248N-K256R, A088T-N109G-N243V-S248N-K256R, A088T-N109G-T158S, A088T-N109G-T158S-N243V-S248N-K256R, A088T-N109G-T158S-N243V-S248N-Q275R, A088T-N109G-T158S-S248N, A088T-N218S-N243V-S248N-K256R-L257G, A088T-N243V-L257G, A088T-S248N, A088T-T158S-K256R, A088T-T158S-N218S-K256R, A088T-T158S-N218S-L257G, A088T-T158S-N218S-L257G, A088T-T158S-N218S-N243V-S248N, A088T-T158S-N218S-N243V-S248N-L257G, A088T-T158S-N218S-Q245K-S248N-K256R, A088T-T158S-N218S-S248N-L257G-Q275K, A088T-T158S-N243V-K256R, A088T-T158S-N243V-K256R-L257G, A088T-T158S-N243V-S248N-K256R, A088T-T158S-S248N, A088T-T158S-S248N-K256R-L257G, A088T-T158S-S248N-L257G, A088T-T158S-S248N-L257G, A098S-G131H-T158S-N218S-N243V-S248N-K256R-L257G, A116T-G131H-K141E-N218S-N243V-S248N-L257G, A116T-G131H-N218S-W241R-N243V-S248N-K256R-L257G, A116T-G131H-N243V, A116T-G131H-T158S-N218S-S248N-K256R, A116T-G131H-V139I-N218S-N243V-S248N, A116T-N218S-S248N-K256R, A116T-T158S-L257G-Q271R, A116T-T158S-N218S-N243V-K256R-L257G, G053S-A088T-N109G-A116T-G131H-T158S-G169S-N218S-S248N-K256R-L257G, G131H-N218S-L257G, G131H-T158S, G131H-T158S-K256R, K256R, L090I-N109G-T158S-N243V, L257G, N109G-A116T-G131H, N109G-A116T-G131H-N243V, N109G-A116T-G131H-T158S-N218S-N243V-S248N-K256R, N109G-A116T-S248N-K256R, N109G-A116T-T158S-N218S-K237R-N243V-S248N, N109G-A116T-T158S-S248N, N109G-G131H-T158S, N109G-G131H-T158S-N243V-K256R-L257G, N109G-G131H-T158S-S248N-Q271R, N109G-N218S-S248N-K256R, N109G-N243V-S248N-L257G, N109G-S248N, N109G-T158S-N218S-N243V-L257G, N109G-T158S-N243V-K256R-L257G, N109G-T158S-N243V-S248N-K256R-L257G, N218S-N243V-S248N-K256R-L257G, S003P-N109G-A116T-G131H-T158S-N218S-K256R, S003P-N109G-G131H-T158S-L257G, S105H-W106G-I107L-I108S-N109A-G110A-I111S-E112N-W113G-A114P, T158S-N218S-S248N-K256R, T158S-N243V-S248N, T158S-S248N, T158S-S248N-K256R-L257G, V004A-A088T-A116T-T158S-N218S, V004A-A088T-G131H-N218S-N243V-S248N-L257G, Y006H-N109G-N218S-N243V-S248N, Y104H-A116T-T158S-S248N, A088T-A116T-G131H-N218S-S248N-L257G, A088T-A116T-G131H-T158S-N218S-N243V-L257G, A088T-A116T-G131H-T158S-N243V-K256R-L257G, A088T-A116T-N218S, A088T-G131D-T158S-N243V-S248N, A088T-G131H-N218S-K237R-K256R-L257G, A088T-G131H-N218S-K256R, A088T-K213N-N243V-S248N-K256R, A088T-K256R-L257G, A088T-N109G-A116T-G131H-D140G-S248N-L257G, A088T-N109G-A116T-G131H-L257G, A088T-N109G-A116T-G131H-N218S-N243V-S248N-K256R-L257G, A088T-N109G-A116T-G131H-T158S-N218S-N243V-S248N, A088T-N109G-A116T-M124I-G131H-T158S-N218S-S248N-L257G, A088T-N109G-A116T-T158S-N218S-N243V-K256R, A088T-N109G-A116T-T158S-N218S-N243V-L257G, A088T-N109G-A116T-T158S-N218S-S248N, A088T-N109G-G131H-K256R-L257G, A088T-N109G-G131H-N218S-N243V-S248N-L257G, A088T-N109G-G131H-N243V-S248N, A088T-N109G-G131H-T158S-L233S-N243V-S248N, A088T-N109G-G131H-T158S-N218S-S248N-K256R, A088T-N109G-L257G, A088T-N109G-N218S-K256R-L257G, A088T-N109G-N218S-S248N-T255K-K256R-L257G, A088T-N109G-S248N-K256R-L257G, A088T-N109G-T158S-N218S-N243V, A088T-N109G-T158S-N218S-N243V-L257G, A088T-N109G-T158S-N218S-N243V-S248N-L257G, A088T-N109G-T158S-N218S-S248N, A088T-S248N-K256R-L257G, A088T-S248N-L257G-I268V, A088T-T158S-N243V-L257G, A088T-T158S-V203I-N218S-K256R-L257G, A088T-Y104H-A116T-G131H-N218S-N243V, A116T-D140G-T158S-N218S-N243V-S248N, A116T-G131H-T158S-K256R-L257G, A116T-G131H-T158S-N243V-S248N, A116T-G157E-T158S-N243V-S248N-K256R, A116T-S248N-K256R, G131H-N218S-S248N, G131H-N243V-K256R, G131H-T158S-N243V-L257G, K256R-L257G, N109G-A116T-G131H-T158S-N218S-N243V-S248N, N109G-A116T-N218S-N243V-L257G, N109G-A116T-N218S-W241R-N243V-S248N-K256R-L257G, N109G-A116T-T158S-N243V, N109G-A116T-T158S-S248N-K256R, N109G-N243V, N109G-T158S-K256R-L257G, N243V, P014L-A015L-L016C-H017T-S018L-Q019K-G020A-Y021T-T022L-G023E, S003P-S248N-L257G, T158S-N218S-N243V-S248N, T158S-S248N-K256R, Y104H-N109G-G131H-N243V-S248N, A088T-A116T-G131H-L257G, A088T-A116T-G131H-N218S-N243V, A088T-A116T-G131H-N218S-S248N-K256R, A088T-A116T-G131H-T158S-N243V-S248N-L257G, A088T-A116T-L257G, A088T-A116T-N218S, A088T-A116T-N218S-N243V-S248N, A088T-A116T-T158S-N218S-S248N, A088T-A116T-T158S-N243V-S248N-K256R, A088T-A138E-N218S-N243V-K256R, A088T-G131H-N218S-N243V-S248N-K256R, A088T-G131H-T158S-S248N-K256R, A088T-N109G-A116T-G131H-N218S-S248N-K256R-L257G, A088T-N109G-A116T-G131H-N243V-S248N-K256R, A088T-N109G-A116T-G131H-T158S-N218S-L257G-I268V, A088T-N109G-A116T-G131H-W241L-S248N-K256R-L257G, A088T-N109G-A116T-N218S-N243V-S248N-K256R, A088T-N109G-A116T-T158S-N243V-K256R-L257G, A088T-N109G-A116T-T158S-N243V-S248N, A088T-N109G-G131H-N218S-L257G, A088T-N109G-G131H-N218S-S248N, A088T-N109G-G131H-T158S-N243V-S248N-L257G, A088T-N109G-N243V-K256R-L257G, A088T-N109G-T158S-S248N-K256R, A088T-N109G-W241R-S248N-K256R, A088T-N218S-S248N-L257G, A088T-T158S-N218S-S248N-K256R, A088T-T158S-N218S-S248N-L257G, A116T-G131H-T158S-N218S-N243V, A116T-N218S-S248N, A116T-N243V-K256R, A116T-T158S, G131H-S248N-K256R, N109G-A116T-G131H-N243V-K256R-L257G, N109G-A116T-I234T-N243V-S248N-K256R-L257G, N109G-A116T-T158S-K256R-L257G, N109G-A116T-T158S-N218S-S248N-K256R, N109G-G131H, N109G-G131H-A137V-T158S-N218S-S248N, N109G-G131H-K141E-L257G, N109G-G131H-T158S-N243V-S248N-L257G, T158S-N218S-N243V-K256R, A088T-A116T-T158S-N243V-K256R-L257G, A088T-N109G-A116T-T158S-K256R-L257G, A088T-N109G-T158S-N218S-S248N, A088T-S248N-L257G, A088T-Y104H-N109G-G131H-A137E-T158S-N218S-N243V-S248N-K256R, G065D-A088T-G131H-N243V-S248N, Y104H-N218S-L257G, A088T-A116T-G131H-V150A-N218S-S248N-L257G, A088T-A116T-T158S-K256R-L257G, A088T-I108T-N109G-G131H-T158S-N218S-S248N-K256R-L257G, A088T-N109G-A116T-T158S-S248N-L257G-Q271P, A088T-N109G-A116T-V148A-N218S-N243V, A088T-Y104H-N109G-A116T-A153S-N218S-N243V-S248N-L257G-N269D, and V004M-A116T-V148A-T158S-N243V-S248N-K256R, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′-v36 and/or BPN-′v3 and/or a PI value of greater than 1.0 compared to BPN′-v36 in a BMI microswatch or egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X001R/T, X002R, X003F/P, X004A/L/M/P, X005S, X006H, X014L, X015L/S, X016C, X017T, X018L, X019K, X020A, X021T, X022L, X023E, X024S, X034S, X053S, X057Q, X065D, X078S, X086L, X088T, X090I, X097A, X098S, X101S, X104H, X105H/P, X106G, X107L/T, X108S/T, X109A/G/S, X110A, X111S, X112N, X113G, X114P/S, X116T, X124I, X128S, X131D/H, X137E/V, X138E/V, X139I, X140G, X141E/R, X143A/F, X144V, X145F/P/T, X146C, X147A/I, X148A, X149A/I/L, X150A, X151S, X152S, X153S, X154A, X155P, X156T, X157E/L, X158M/S, X159E, X160E, X161L, X169S, X182F, X203I, X204F, X207L, X211V, X212D, X213N, X216S, X218S/T, X223G, X231V, X232S, X233S, X234T, X235P, X236F/P, X237N/R, X240H, X241L/R, X243F/V, X245K, X248N, X251K, X255K, X256R, X257G, X260F, X267M, X268V, X269D/S, X271H/K/P/R, X272G/V, X273T, X274D/L/T/V, and X275K/R/S, and optionally at least one substitution selected from the group of A001R/T, Q002R, S003F/P, V004A/L/M/P, P005S, Y006H, P014L, A015L/S, L016C, H017T, S018L, Q019K, G020A, Y021T, T022L, G023E, G024S, G034S, G053S, P057Q, G065D, N078S, P086L, A088T, L090I, G097A, A098S, N101S, Y104H, S105H/P, W106G, I107L/T, I108S/T, N109A/G/S, G110A, I111S, E112N, W113G, A114P/S, A116T, M124I, A128S, G131D/H, A137E/V, A138E/V, V139I, D140G, K141E/R, V143A/F, A144V, S145F/P/T, G146C, V147A/I, V148A, V149A/I/L, V150A, A151S, A152S, A153S, G154A, N155P, E156T, G157E/L, T158M/S, S159E, G160E, S161L, G169S, S182F, V203I, S204F, S207L, G211V, N212D, K213N, A216S, N218S/T, A223G, A231V, A232S, L233S, I234T, L235P, S236F/P, K237N/R, N240H, W241L/R, N243F/V, Q245K, S248N, E251K, T255K, K256R, L257G, S260F, L267M, I268V, N269D/S, Q271H/K/P/R, A272G/V, A273T, A274D/L/T/V, Q275K/R/S, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 10 Construction and Cleaning Performance of Additional Variants of BPN′-v36

The DNA from the site evaluation libraries of the BPN′-v36 (described in Example 7) was further mutagenized by error-prone PCR. These libraries were amplified with primers P4973 and P4950 (described in Example 7) using Taq DNA polymerase (Promega). Each PCR amplification reaction contained 30 pmol of each primer, 100 ng of the template DNA (SELs of the BPN′-v36) and various amount of MnCl₂. The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 2 min. The DNA fragment was gel-purified by the QIAGEN® gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ partial opt vector that also was digested with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification in an Illustra Templiphi kit according to the manufacturer's recommendation (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C.

About 500,000 clones were pre-screened on skim milk plates. Very few of them formed halos (i.e., indicative of the presence of functional protease). Colonies with halos were picked, inoculated in 150 μl of LB media containing 10 μg/mL neomycin and sequenced (Quintara). Sequences of these clones were analyzed by looking for combination of mutations, which occurred in this pool multiple times and might provide performance benefits. In order to assess the performance of these mutation combinations, double mutants were created in the BPN′-v36 background by PCR fusion as described below. For this purpose, two or three partially overlapping fragments were amplified by mutagenic primers. Primer combinations used to generate the respective variants are shown in Table 10-1 and primer sequences are shown in Table 10-2.

TABLE 10-1 Primers Pairs Used to Amplify Fragments Variant Mutation 1 Mutation 2 Fragment 1 Fragment 2 Fragment 3 1 A45S S236Y P4974, P6644, P6647 P6646, P4976 P6645 2 A45S S236G P4974, P6644, P6649 P6648, P4976 P6645 3 I115T S183T P4974, P6650, P6655 P6654, P4976 P6651 4 I115V N184Y P4974, P6652, P6657 P6656, P4976 P6653 5 I31T S37P P4974, P6658, P4976 P6659 6 I31T I35L P4974, P6660, P4976 P6661 7 I31V S38W P4974, P6662, P4976 P6663 8 N25K P129R P4974, P6664, P6667 P6666, P4976 P6665 9 N25K P129K P4974, P6664, P6669 P6668, P4976 P6665 10 P14T S37T P4974, P6670, P6673 P6672, P4976 P6671 11 P5L Q217K P4974, P6678, P6681 P6680, P4976 P6679 12 P5L Q217G P4974, P6678, P6683 P6682, P4976 P6679 13 Q10L S37P P4974, P6684, P6675 P6674, P4976 P6685 14 Q10R S37T P4974, P6686, P6673 P6672, P4976 P6687 15 S37P T254S P4974, P6674, P6689 P6688, P4976 P6675 16 N25K S37P P4974, P6664, P6675 P6674, P4976 P6665 17 G24A S37W P4974, P6690, P6677 P6676, P4976 P6691 18 N25K P129R P4974, P6664, P6667 P6666, P4976 P6665 20 S161P S162L P4974, P6694, P4976 P6695 21 S161P T253A P4974, P6692, P6701 P6700, P4976 P6693 22 S161P S260P P4974, P6692, P6703 P6702, P4976 P6693 23 S162L D181H P4974, P6696, P6711 P6710, P4976 P6697 24 S162L D181G P4974, P6696, P6713 P6712, P4976 P6697 25 S18F S162L P4974, P6714, P6697 P6696, P4976 P6715 26 S18T S162P P4974, P6716, P6699 P6698, P4976 P6717 27 S18P D120N P4974, P6718, P6727 P6726, P4976 P6719 28 S18Y K213R P4974, P6720, P6729 P6728, P4976 P6721 29 S18L Y21S P4974, P6730, P4976 P6731 30 S18T Y21N P4974, P6732, P4976 P6733 31 S9T K141F P4974, P6734, P6737 P6736, P4976 P6635 32 S9T K141R P4974, P6734, P6739 P6738, P4976 P6635 33 Q19L S260N P4974, P6724, P6705 P6704, P4976 P6725 34 Q19L S260P P4974, P6724, P6703 P6702, P4976 P6725 35 N61S S260P P4974, P6740, P6703 P6702, P4976 P6741 36 N61D S260I P4974, P6742, P6707 P6706, P4976 P6743 37 T253A S260P P4974, P6700, P6703 P6702, P4976 P6701 38 A134T S260G P4974, P6744, P6709 P6708, P4976 P6745 39 A133V S260N P4974, P6746, P6705 P6704, P4976 P6648

TABLE 10-2 Primer Sequences Used for Generation of Double Mutants of BPN′-v36 Primer Name SEQ ID NO: P6644 SEQ ID NO: 64 P6645 SEQ ID NO: 65 P6646 SEQ ID NO: 66 P6647 SEQ ID NO: 67 P6648 SEQ ID NO: 68 P6649 SEQ ID NO: 69 P6650 SEQ ID NO: 70 P6651 SEQ ID NO: 71 P6652 SEQ ID NO: 72 P6653 SEQ ID NO: 73 P6654 SEQ ID NO: 74 P6655 SEQ ID NO: 75 P6656 SEQ ID NO: 76 P6657 SEQ ID NO: 77 P6658 SEQ ID NO: 78 P6659 SEQ ID NO: 79 P6660 SEQ ID NO: 80 P6661 SEQ ID NO: 81 P6662 SEQ ID NO: 82 P6663 SEQ ID NO: 83 P6664 SEQ ID NO: 84 P6665 SEQ ID NO: 85 P6666 SEQ ID NO: 86 P6667 SEQ ID NO: 87 P6668 SEQ ID NO: 88 P6669 SEQ ID NO: 89 P6670 SEQ ID NO: 90 P6671 SEQ ID NO: 91 P6672 SEQ ID NO: 92 P6673 SEQ ID NO: 93 P6674 SEQ ID NO: 94 P6675 SEQ ID NO: 95 P6676 SEQ ID NO: 96 P6677 SEQ ID NO: 97 P6678 SEQ ID NO: 98 P6679 SEQ ID NO: 99 P6680 SEQ ID NO: 100 P6681 SEQ ID NO: 101 P6682 SEQ ID NO: 102 P6683 SEQ ID NO: 103 P6684 SEQ ID NO: 104 P6685 SEQ ID NO: 105 P6686 SEQ ID NO: 106 P6687 SEQ ID NO: 107 P6688 SEQ ID NO: 108 P6689 SEQ ID NO: 109 P6690 SEQ ID NO: 110 P6691 SEQ ID NO: 111 P6692 SEQ ID NO: 112 P6693 SEQ ID NO: 113 P6694 SEQ ID NO: 114 P6695 SEQ ID NO: 115 P6696 SEQ ID NO: 116 P6697 SEQ ID NO: 117 P6698 SEQ ID NO: 118 P6699 SEQ ID NO: 119 P6700 SEQ ID NO: 120 P6701 SEQ ID NO: 121 P6702 SEQ ID NO: 122 P6703 SEQ ID NO: 123 P6704 SEQ ID NO: 124 P6705 SEQ ID NO: 125 P6706 SEQ ID NO: 126 P6707 SEQ ID NO: 127 P6708 SEQ ID NO: 128 P6709 SEQ ID NO: 129 P6710 SEQ ID NO: 130 P6711 SEQ ID NO: 131 P6712 SEQ ID NO: 132 P6713 SEQ ID NO: 133 P6714 SEQ ID NO: 134 P6715 SEQ ID NO: 135 P6716 SEQ ID NO: 136 P6717 SEQ ID NO: 137 P6718 SEQ ID NO: 138 P6719 SEQ ID NO: 139 P6720 SEQ ID NO: 140 P6721 SEQ ID NO: 141 P6722 SEQ ID NO: 142 P6723 SEQ ID NO: 143 P6724 SEQ ID NO: 144 P6725 SEQ ID NO: 145 P6726 SEQ ID NO: 146 P6727 SEQ ID NO: 147 P6728 SEQ ID NO: 148 P6729 SEQ ID NO: 149 P6730 SEQ ID NO: 150 P6731 SEQ ID NO: 151 P6732 SEQ ID NO: 152 P6733 SEQ ID NO: 153 P6734 SEQ ID NO: 154 P6735 SEQ ID NO: 155 P6736 SEQ ID NO: 156 P6737 SEQ ID NO: 157 P6738 SEQ ID NO: 158 P6739 SEQ ID NO: 159 P6740 SEQ ID NO: 160 P6741 SEQ ID NO: 161 P6742 SEQ ID NO: 162 P6743 SEQ ID NO: 163 P6744 SEQ ID NO: 164 P6745 SEQ ID NO: 165 P6746 SEQ ID NO: 166 P6747 SEQ ID NO: 167

Each PCR amplification reaction contained 30 pmol of each primer and 100 ng of the BPN′-v36 parent template DNA (plasmid pHPLT-BPN′-v36) (see FIG. 4). Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 40 sec. Following amplification, the 5′ and 3′ gene fragments were gel-purified by the QIAGEN® gel-band purification kit, mixed (50 ng of each fragment), mixed and amplified by PCR once again using the primers P4973 and P4950 to generate the full-length gene fragment. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min The full-length DNA fragment was gel-purified by the QIAGEN® gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ partial opt vector that also was digested with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification in an Illustra Templiphi kit according to the manufacturer's recommendation (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, the colonies with halos were inoculated in 150 μl of LB media containing 10 μg/mL neomycin. The next day, the cultures were either frozen with 15% glycerol or grown in MBD medium for biochemical analysis as described in Example 2.

The variants were tested for cleaning performance using BMI microswatch assay in Detergent Composition 4 at 16° C. and pH 8, BMI microswatch assay in Detergent Composition 4 at 16° C. and pH 7, and Egg microswatch assay in Detergent Composition 4 at 16° C. and pH 8. Protein content was determined using TCA assay. All assays were performed as described in Example 1 and Performance Indices were calculated relative to BPN′-v36 (i.e., BPN′-S24G-S53G-S78N-S101N-G128A-Y217Q).

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A133V-S260N, N061S-S260P, P014T-S037T, S009T-K141F, S009T-K141R, S018F-S162L, S018L-Y021S, S018P-D120N, S018T-S162P, S018T-Y021N, S018Y-K213R, S161P-S162L, S161P-S260P, and T253A-S260P, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A134T-S260G, I115V-N184Y, N025K-S037P, Q010L-S037P, Q019L-S260N, Q019L-S260P, S037P-T254S, and S161P-T253A, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A045S-S236G, G024A-S037W, I031V-S038W, N061D-S260I, Q010R-S037T, I115T-S183T, N025K-P129K, N025K-P129R, A045S-S236Y, and S162L-D181H, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 7 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of S018F-S162L, S018P-D120N, P014T-S037T, S009T-K141R, and S161P-S162L, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 7 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, N061S-S260P, Q010L-S037P, S009T-K141F, S018L-Y021S, S018T-S162P, S018T-Y021N, S018Y-K213R, S037P-T254S, S161P-S260P, S161P-T253A, and T253A-S260P, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′ variants were determined to have a PI value of about 0.9 relative to BPN′-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 7 and 16° C.: BPN′ amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of A133V-S260N, A134T-S260G, I115T-S183T, I115V-N184Y, N061D-S260I, Q019L-S260N, and Q019L-S260P, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN′-v3, and/or an enhanced proteolytic activity compared to BPN′ in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 7 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A045S-S236G, G024A-S037W, Q010R-S037T, A045S-S236Y, I031V-S038W, N025K-S037P, S162L-D181H, and N025K-P129R, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of I031V-S038W, P014T-S037T, S018F-S162L, S018P-D120N, and S162L-D181H, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v36, A133V-S260N, A134T-S260G, G024A-S037W, I1115V-N184Y, N025K-P129K, N025K-P129R, N061D-S260I, Q019L-S260P, S009T-K141F, S009T-K141R, S018L-Y021S, S018T-S162P, S018T-Y021N, S018Y-K213R, S161P-S162L, S161P-T253A, and T253A-S260P, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of 1.0 relative to BPN′-v3, and a PI value of 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.8 and equal to or less than 0.9 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A045S-S236G, A045S-S236Y, I115T-S183T, N025K-S037P, N061S-S260P, Q010L-S037P, Q010R-S037T, Q019L-S260N, S037P-T254S, and S161P-S260P, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′-v36 and/or a PI value of greater than 1.0 compared to BPN′-v36 in a BMI or egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution comprising at least one substitution selected from the group of X009T, X010L/R, X014T, X018F/L/P/T/Y, X019L, X021N/S, X024A, X025K, X031V, X037P/T/W, X038W, X045S, X061D/S, X115T/V, X120N, X129K/R, X133V, X134T, X141F/R, X161P, X162L/P, X181H, X183T, X184Y, X213R, X236G/Y, X253A, X254S, and X260G/I/N/P, and optionally at least one substitution selected from the group of S009T, Q010L/R, P014T, S018F/L/P/T/Y, Q019L, Y021N/S, G024A, N025K, I031V, S037P/T/W, S038W, A045S, N061D/S, I115T/V, D120N, P129K/R, A133V, A134T, K141F/R, S161P, S162L/P, D181H, S183T, N184Y, K213R, S236G/Y, T253A, T254S, and S260G/I/N/P, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.

Example 11 1. Generation of Combinatorial Libraries FS1-FS3

The pHPLT-BPN′-v3 plasmid containing the BPN′ expression cassette served as template DNA (parent plasmid) for cloning. Three separate combinatorial libraries (FS1, FS2, and FS3) were synthesized by DNA2.0, and were delivered as individual ligation reactions. A list of libraries and possible substitutions are shown in Table 11-1. The libraries were designed to allow the incorporation of either the wild type residues or the substitutions at each site described in Table 11-1.

For efficient transformation of B. subtilis, the DNA from the ligation reaction was amplified by rolling circle amplification (RCA) using the Illustra Templiphi kit (GE Healthcare). Reactions were performed according to the manufacturer's protocol. One microliter of ten-fold diluted amplified DNA was used to transform 50 μL of competent B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). The transformation mixture was shaken at 37° C. for 1 hour. Ten microliter aliquots of the transformation mixture were plated on skim milk (1.6%) Luria Agar plates supplemented with 10 μg/ml of neomycin (Teknova).

Transformants were picked into microtiter plates containing 125-150 μl Luria broth medium supplemented with 10 μg/ml neomycin. Plates were grown overnight at 37° C. with 250-300 rpm shaking and 70-80% humidity using Enzyscreen lids for microtiter plates (Enzyscreen). Between 7 and 10 microliters from the overnight culture plate were used to inoculate a new microtiter plate containing 190 μl of MBD medium (a MOPS based defined medium) with 10 μg/ml neomycin. MBD medium was prepared essentially as known in the art (see Neidhardt et al., J. Bacteriol. 119:736-747 (1974)), except that NH₄Cl, FeSO₄, and CaCl₂ were omitted from the base medium, 3 mM K₂HPO₄ was used, and the base medium was supplemented with 60 mM urea, and 100 ml of a solution made of 210 g/L glucose, and 350 g/L maltodextrin. The micronutrients were made up as a 100× stock solution containing in one liter, 400 mg FeSO₄.7H₂O, 100 mg MnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100 mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1 M CaCl₂, and 10 ml of 0.5 M sodium citrate. The MBD medium containing microtiter plates were grown for 60-70 hours at 37° C., 250-300 rpm, and 70-80% humidity using Enzyscreen lids (Enzyscreen) for determining protein expression. The next day, cultures were filtered through a micro-filter plate (0.22 μm; Millipore) and the resulting filtrate was used for biochemical analysis.

TABLE 11-1 Possible Substitutions for Combinatorial Libraries FS1-FS3 FS1 FS2 FS3 1 G102A A97G N61E 2 S130P A128G P129E 3 T55P N123G K213L 4 V203Y G102A S145D 5 N61P L126V Q275E 6 S101N G100N P40E 7 S53G N62Q S159K 8 S78N M124I S24R 9 S87T-A88L- N61P A144K S89G 10 S24G-N25G S130P N240K 11 L75S-N76Y P129S P239R

2. Generation of Variants to Improve BPN′ Stability

To improve BPN′ stability, variants were constructed using either parent molecules pHPLT-BPN′ G97A-G128A-Y217Q-S87D or pHPLT-BPN′ G97A-G128A-Y217Q-P40E, both synthesized by Gene Oracle, or parent molecules pHPLT-BPN′ G97A-G128A-Y217Q-S78N and pHPLT-partial opt FNA (B. amyloliquefaciens subtilisin BPN′-Y217L) synthesized by GeneArt.

The information listed in Tables 11-2 and 11-3 summarizes the parent molecule used, the mutations added, and the primers used to construct variants provided herein.

TABLE 11-2 Primer Sequences Used for the Generation of BPN′ Stability Mutants Primer Name SEQ ID NO: p31 169 p32 170 p25 171 p26 172 p33 173 p34 174 p29 175 p30 176 p27 177 p28 178 p7 179 p8 180 p21 181 p22 182 p11 183 p12 184 p13 185 p14 186 p15 187 p16 188 p17 189 p18 190

TABLE 11-3 Templates Used, Mutations to be Incorporated, and Primers Used in the Generation of Variants for Improved Stability Mutation to Primers Parent Molecules (Templates) Incorporate Used 1 pHPLT-BPN′ G97A-G128A-Y217Q- S78N p31, p32 S87D 2 pHPLT-BPN′ G97A-G128A-Y217Q- P40E p25, p26 S78N 3 pHPLT-BPN′ G97A-G128A-Y217Q- S87D p33, p34 P40E 4 pHPLT-BPN′ G97A-G128A-Y217Q- P40E, p25, p33 S78N S87D 5 pHPLT-BPN′ G97A-G128A-Y217Q- N76D p29, p30 S87D 6 pHPLT-BPN′ G97A-G128A-Y217Q- N76D, p27, p28 S87D S78N 7 pHPLT-partial opt FNA S78N p7, p8 8 pHPLT-partial opt FNA S87D p21, p22 9 pHPLT-partial opt FNA P40E p11, p12 10 pHPLT-partial opt FNA K213N p13, p14 11 pHPLT-partial opt FNA T22V p15, p16 12 pHPLT-partial opt FNA Q206E p17, p18 13 pHPLT-partial opt FNA P40E, p11, p21, S87D, p7 S78N

Bacillus subtilis strains expressing plasmids were streaked onto 1.6% skim milk plates containing 10 ppm neomycin and grown overnight at 37° C. Single colonies from the plates were grown overnight at 37° C. with shaking at 250 rpm in 5 mL Luria broth containing 10 ppm neomycin. Plasmids were isolated using the QIAGEN® Miniprep kit protocol adding 1 microliter of Ready Lyse lysozyme (Epicentre) for 15 minutes at 37° C. in buffer P1 to aid in cell lysis. The plasmids were sequenced to ensure correct DNA sequences before proceeding. The plasmids were methylated using NEB's Dam Methylase Kit in a reaction containing 77.5 μL water+10 μL Buffer 10×+0.25 μL SAM+2 μL DAM methylase+10 μL miniprep DNA (˜150 ng/μL) at 37° C. overnight. The methylated plasmid DNA was purified using a DNA Clean and Concentrator Kit (Zymo) or with a QIAGEN® PCR purification kit. Multi-Site QUIKCHANGE® mutagenesis reactions were set up for each of the DNA templates in a reaction mix containing 2.5 μL Buffer 5×+0.75 μL Quik Solution+0.5 μL primer 1 (25 μM)+0.5 μL primer 2 (25 μM)+1.5 μL dNTP's+1 μL enzyme blend+16.25 μL H₂O+2 μL DNA. The PCR program used was: 95° C. for 1 min; (95° C. for 1 min, 53° C. for 1 min, 65° C. for 10 min)×29 cycles; 65° C. for 10 min, 4° C. hold. In all reactions, PCR was performed using a MJ Research PTC-200 Peltier thermal cycler. The parental DNA from the PCR samples was removed by addition of 1 μL of DpnI to QUIKCHANGE® mutagenesis kit reactions at 37° C. overnight. To increase the transformation frequency, the DpnI digested reactions were amplified using rolling circle amplification (RCA) using the Illustra TempliPhi kit according to the manufacturer's protocol. B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) were transformed with 1 μL each of the RCA reaction and the transformed cells were plated onto LA+1.6% skim milk plates containing 10 ppm neomycin and incubated at 37° C. overnight. Colonies from overnight growth were selected to perform colony PCR for sequencing using “puReTaq Ready-To-Go PCR Beads” (Amersham). The PCR and sequencing primers used were pHPLT F1 (SEQ ID NO:54) and pHPLT seq R1 (SEQ ID NO:55). Clones with appropriate sequences were frozen. The BPN′ variant proteins were produced by growing the B. subtilis transformants in 96 well microtiter plates at 37° C. for 68 hours in a MOPS based medium containing urea.

3. Generation of BPN′ Variants Derived from Five Different Parent Plasmids

BPN′ variants were constructed using a total of five different templates: BPN′-v3 (G97A-G128A-Y217Q), BPN′-v4 (G97A-N123G-Y217Q), BPN′ variant 8, (S87D-G97A-N109D-G128A-S188D-S248R-Y217Q), BPN′ variant 16 (S87D-G97A-N109D-G128A-S188D-Y217Q), and BPN′ variant 21 (S87R-G97A-N109R-G128A-S188R-Y217Q-S248R) as shown in Table 11-4. The generation of BPN′-v4 and BPN′-v3 are described in PCT App. No. PCT/US09/46066 (WO 09/149144), filed on Jun. 3, 2009, hereby incorporated herein by reference for such description. BPN′ variants 8, 16, 21 were synthesized by Gene Oracle and served as parent plasmids to build additional variants. All variants were generated using QUIKCHANGE® mutagenesis kits, except two (variants 5 and 33), which were generated using fusion PCR as described below. Primers (listed in Table 11-5) for the generation of variants were synthesized at Integrated DNA Technologies. The mutations introduced (shown in bold) and the primers and template used are shown in Table 11-4.

TABLE 11-4 Mutations Introduced (bold) & Parent Plasmids Used to Generate BPN′ Variants Variant Variants Constructed Parent Plasmid Primers Used 1 G97A-N123C-Y217Q G97A-N123G-Y217Q N123C f, N123C r 2 N76D-G97A-G128A- G97A-G128A-Y217Q N76D f, N76D r Y217Q 3 G97A-N109D-G128A- G97A-G128A-Y217Q N109D f1, N109D r Y217Q 4 G97A-G128A-S188D- G97A-G128A-Y217Q S188D f, S188D r Y217Q 5 G97A-G128A-S248D- G97A-G128A-Y217Q pHPLT F1, S248D Y217Q forfus, pHPLT R1, S248D revfus 6 G97A-G128A-S188D- G97A-G128A-Y217Q S188D f, S248R f1 S248R-Y217Q 7 G97A-N109D-G128A- S87D-G97A-N109D- D87S f, D87S r S188D-S248R-Y217Q G128A-S188D-S248R- Y217Q 8 S87D-G97A-N109D- Synthesized by Gene Synthesized by Gene G128A-S188D-S248R- Oracle Oracle Y217Q 9 S87R-G97A-G128A- S87D-G97A-N109D- S87R f, S248D f1, S188D-S248D-Y217Q G128A-S188D-Y217Q D109N f 10 S87R-G97A-N109D- S87D-G97A-N109D- R248Sfor, R248Srev G128A-S188D-Y217Q G128A-S188D-S248R- Y217Q 11 G97A-N109D-G128A- G97A-G128A-Y217Q N109D f2, S188R f S188R-Y217Q 12 S87R-G97A-N109D- S87D-G97A-N109D- S87R f, S87R r G128A-S188D-Y217Q- G128A-S188D-S248R- S248R Y217Q 13 G97A-N109D-G128A- G97A-G128A-Y217Q N109D f2, S248R f2 Y217Q-S248R 14 S87D-G97A-G128A- G97A-G128A-Y217Q S87D f, S248R f1 Y217Q-S248R 15 S87D-G97A-N109D- S87D-G97A-N109D- S248D f1, S248D r G128A-S188D-Y217Q- G128A-S188D-Y217Q S248D 16 S87D-G97A-N109D- Synthesized by Gene Synthesized by Gene G128A-S188D-Y217Q Oracle Oracle 17 G97A-N109D-G128A- G97A-G128A-Y217Q N109D f2, S248D f2 Y217Q-S248D 18 S87R-G97A-G128A- G97A-G128A-Y217Q S87R f, S87R r Y217Q 19 S87R-G97A-G128A- G97A-G128A-Y217Q S87R f, S248R f1 Y217Q-S248R 20 S87R-G97A-G128A- S87R-G97A-N109R- D109N f, D109N r S188R-Y217Q-S248R G128A-S188R- Y217Q-S248R 21 S87R-G97A-N109R- Synthesized by Gene Synthesized by Gene G128A-S188R-Y217Q- Oracle Oracle S248R 22 G97A-G102A-G128A- G97A-G128A-Y217Q G102A f, G102A r Y217Q 23 G97A-G128A-S130P- G97A-G128A-Y217Q S130P f, S130P r Y217Q 24 G97A-S101N-G128A- G97A-G128A-Y217Q S101N f, S101N r Y217Q 25 G97A-G100N-G128A- G97A-G128A-Y217Q G100N f, G100N r Y217Q 26 N61P-G97A-G128A- G97A-G128A-Y217Q N61P f, N61P r Y217Q 27 G97A-G128A-A187D- G97A-G128A-Y217Q A187D f, A187D r Y217Q 28 G97A-G128A-F189D- G97A-G128A-Y217Q f189D f, f189D r Y217Q 29 G97A-G128A-A137V- G97A-G128A-Y217Q A137V f, A137V r Y217Q 30 S63T-G97A-G128A- G97A-G128A-Y217Q S63T f, S63T r Y217Q 31 G97A-Q103N-G128A- G97A-G128A-Y217Q Q103N f, Q103N r Y217Q 32 N62D-G97A-G128A- G97A-G128A-Y217Q N62D f, N62D r Y217Q 33 G97A-G100E-G128A- G97A-G128A-Y217Q G100E Fsfor, pHPLT Y217Q F1, G100E Fsrev, pHPLT R1

Generation of BPN′ Variants Via QUIKCHANGE® Mutagenesis

Bacillus subtilis strains containing plasmids expressing BPN′-v3, BPN′-v4, BPN′ variant 8, BPN′ variant 16, and BPN′ variant 21 were streaked onto 1.6% skim milk plates containing 10 ppm neomycin and grown overnight at 37° C. Single colonies from the plates were grown overnight at 37° C. with shaking at 250 rpm in 5 mL Luria broth containing 10 ppm neomycin. Plasmids expressing BPN′-v3, BPN′-v4, BPN′ variant 8, BPN′ variant 16, and BPN′ variant 21 were isolated using QIAGEN® Miniprep kit protocol except following cell lysis, 1 microliter of Ready Lyse lysozyme was added and incubated for 15 minutes at 37° C. The plasmids were methylated using NEB's Dam Methylase Kit in a reaction containing 77.75 μL H₂O+10 μL Buffer 10×+0.25 μL SAM+2 μL DAM methylase+10 μL miniprep DNA at 37° C. overnight. The methylated DNA was purified using the QIAGEN® PCR purification kit. Variants 14, 18, and 19 listed in Table 11-4 were generated using QUIKCHANGE LIGHTNING™ Multi Site-Directed Mutagenesis kits (Stratagene) in a reaction mix containing 2.5 μL Buffer 5×+0.75 μL Quik Solution+0.5 μL primer 1 (25 μM)+0.5 μL primer 2 (25 μM)+1.5 μL dNTP's+1 μL enzyme blend+16.25 μL H₂O+2 μL DNA. The PCR program used was as follows: 95° C. for 2 min; (95° C. for 20 sec, 55° C. for 30 sec, 64° C. for 2 min 30 sec)×29 cycles; 64° C. for 5 min, 4° C. hold.

The remaining variants were created using QUIKCHANGE® Multi Site-Directed Mutagenesis kits in a reaction mix containing 2.5 μL Buffer 5×+0.75 μL Quik Solution+0.5 μL primer 1 (25 μM)+0.5 μL primer 2 (25 μM)+1.5 μL dNTP's+1 μL enzyme blend+16.25 μL H₂O+2 μL DNA. The PCR program used was as follows: 95° C. for 1 min; (95° C. for 1 min, 53° C. for 1 min, 65° C. for 10 min)×29 cycles; 65° C. for 10 min, 4° C. hold. In all reactions, PCR was performed using a MJ Research PTC-200 Peltier thermal cycler. The primers used for the Quik-Change reactions are provided in Table 11-5. The primers are shown in primer sequence (5′ to 3′).

TABLE 11-5 Primers Used for Quik-Change Reactions Primer Name SEQ ID NO: N76D f 191 N76D r 192 S87D f 193 S87D r 194 G102A f 195 G102A r 196 S130P f 197 S130P r 198 S101N f 199 S101N r 200 G100N f 201 G100N r 202 N61P f 203 N61P r 204 A187D f 205 A187D r 206 F189D f 207 F189D r 208 A137V f 209 A137V r 210 S63T f 211 S63T r 212 Q103N f 213 Q103N r 214 N62D f 215 N62D r 216 N109D f1 217 N109D r 218 S188D f 219 S188D r 220 S248R f1 221 S87R f 222 S87R r 223 S248D f1 224 S248D r 225 D87S f 226 D87S r 227 D109N f 228 D109N r 229 N109D f2 230 S248R f2 231 S248D f2 232 S188R f 233 QC FUSION_For1 234 QC FUSION_Rev1 235 S248D forfus 236 S248D revfus 237 R248Sfor 238 R248S rev 239 G100E_Fsfor 240 G100E_Fsrev 241 N123C f 242 N123C r 243 pHpLT R1 244

The parental DNA from the PCR samples was removed by addition of 1 μL of DpnI to Quik-Change reactions at 37° C. overnight. One micro-liter of the DpnI-digested reactions were amplified using rolling circle amplification (RCA) using the Illustra TempliPhi kit according to the manufacturer's protocol. B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) were transformed with 1 μL each of the RCA reaction and the transformed cells were plated onto LA+1.6% skim milk plates containing 10 ppm neomycin and incubated at 37° C. overnight. Colonies from overnight growth were selected to perform colony PCR using “puReTaq Ready-To-Go PCR Beads” (Amersham). The PCR primers used were pHPLT F1 (SEQ ID NO:54) and pHPLT seq R1 (SEQ ID NO:55). Clones with appropriate sequences were frozen. BPN′ variants were expressed by growing the B. subtilis transformants in 96 well microtiter plates at 37° C. for 68 hours in a MOPS based medium containing urea.

Generation of BPN′ Variants Via Fusion PCR

Variants 5 and 33 were generated using Fusion PCR, with fragments amplified from template pHPLT-BPN′-v3. The PCR primers used to generate these variants are included in Table 11-5. For Variant 5, a 5′ fragment of the BPN′ gene was amplified using forward primer pHPLT F1(SEQ ID NO:54), and reverse primer S248D revfus. The 3′ fragment of the BPN′ gene was amplified using the forward primer S248D forfus containing the mutation of interest and the reverse primer pHPLT RE The two products contained 20 bp of overlapping sequence, and were fused by combining 1 μL of each fragment and fusion primers QC FUSION_For1 and QC FUSION_Rev1 in a final PCR reaction. All PCR reactions were performed using standard conditions of the Herculase II PCR Kit (Stratagene). The PCR mix contained 1 μL DNA polymerase, 1 μL plasmid DNA (or fragment DNA for fusion), 0.5 μL dNTP's, 1.25 μL 25 μM forward primer, 1.25 μL 25 μM reverse primer, 10 μL Buffer 5×, 35 μL H₂O and the PCR program used was as follows: 95° C. for 2 min, (95° C. for 30 sec, 55° C. for 30 sec, 72° C. for “X” sec) for 29 cycles, 72° C. for 1 min, 4° C. hold (the “X” is 15 seconds per 1kB of DNA to amplify).

For Variant 33, a 5′ fragment of the BPN′ gene was amplified using the template pHPLT-BPN′-v3 and primers pHPLT F1 (SEQ ID NO:54), and G100E_Fsrev. The 3′ fragment that contained the variant was amplified using primers G100E_Fsfor and pHPLT RE The two products contained 20 bp of overlapping sequence, and were fused by combining 1 μl of each fragment and fusion primers QC FUSION_For1 and QC FUSION_Rev1 in a final PCR reaction. The PCR conditions were the same as listed above.

The two fusion products were purified using a QIAGEN® PCR purification column with conditions provided by the manufacturer, and digested overnight using Bgl I and HindIII enzymes. The plasmid pHPLT-BPN′ partial opt was digested using the same enzymes and the vector band was gel extracted and purified over a QIAGEN® gel purification column using the manufacturer's recommendations. The restriction enzyme mix contained: 10 μL purified DNA, 5 μL Roche Buffer B, 0.5 μL HindIII, 0.5 μL Bgl I, 34 μL H20 and the reactions were carried out at 37° C. for 8 hours followed by 65° C. for 20 min. The digest was purified using a QIAGEN® PCR purification column and ligated to the cut vector backbone overnight at 16° C. using the Mighty Mix Ligase kit (Tekara). Following incubation, 1 μL of the ligation mix was amplified using the Illustra TempliPhi kit.

For the amplification reaction, 1 μL of the ligation reaction mix was mixed with 5 μL of sample buffer from the TempliPhi kit and heated for 3 minutes at 95° C. to denature the DNA. The reaction was placed on ice to cool for 2 minutes and then spun down briefly. Five microliters of reaction buffer and 0.2 μL of phi29 polymerase from the TempliPhi kit were added, and the reactions were incubated at 30° C. in an MJ Research PCR machine for 4 hours. The phi29 enzyme was heat inactivated in the reactions by incubation at 65° C. for 10 min in the PCR machine. Bacillus subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) were transformed using 1 μL of the reaction mix and the transformants were grown overnight at 37° C. on 1.6% skim milk plates containing 10 ppm neomycin. Transformants were selected to perform colony PCR and sequencing using “puReTaq Ready-To-Go PCR Beads” (Amersham) and primers pHPLT F1 (SEQ ID NO:54) and pHPLT seqR1 (SEQ ID NO:55).

4. Generation of BPN′ Variants from Libraries RCL4-RCL7

RCL4 Library

“RCL4” refers to a group of site saturation libraries created by PCR fusion that simultaneously randomize three contiguous codons in the BPN′-v3-encoding (BPN′-G97A-G128A-Y217Q) gene. The amino acid positions corresponding to the three mutated codons in each library are provided in Table 11-6. Two partially overlapping, complementary mutagenic primers, each containing three degenerate codons were used to introduce mutations within each library as shown in Table 11-6 below. Only the first two nucleotides of each degenerate codon (NNX, N=A, C, T, or G and X is unchanged nucleotide) of interest were mutated in each primer (Table 11-6).

To create each library, two PCR reactions were carried out using either the common 3′ gene-flanking primer (P4976, CCTCTCGGTTATGAGTTAGTTC; (SEQ ID NO:61)) and mutagenic primer, or the common 5′gene-flanking primer (P4974, GCCTCACATTTGTGCCACCTA; (SEQ ID NO:60)) and mutagenic primer as shown for each library in Table 11-6. These PCR reactions generated two PCR fragments, one encoding the 5′ half of the mutant BPN′-v3 gene (5′ gene fragment) and the other encoding the 3′ half of the mutant BPN′-v3 gene (3′ gene fragment). Each PCR amplification reaction contained 30 pmol of each primer and 100 ng of the BPN′-v3 parent template DNA (plasmid pHPLT-BPN′-v3) (see FIG. 1). Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 40 sec. Following amplification, the 5′ and 3′ gene fragments were gel-purified by the QIAGEN® gel-band purification kit, mixed (50 ng of each fragment) and amplified by PCR once again using the primers P4973 (AAAGGATCCTAATCGGCGCTTTTC; SEQ ID NO:62) and P4950 (CTTGTCTCCAAGCTTAAAATAAAA; SEQ ID NO:63) to generate the full-length gene fragment. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min. The full-length DNA fragment was gel-purified by the QIAGEN® gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ partial opt vector that also was digested with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification in an Illustra Templiphi kit according to the manufacturer's recommendation (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, the colonies with halos were inoculated in 120 μl of LB media containing 10 μg/mL neomycin.

TABLE 11-6 List of Primers Used to Create RCL4 Libraries Mutated residues Common in flanking Mutagenic Library BPN′- primer primer # v3 Gene fragment names names SEQ ID NO: 1 5-7 3′ P4976 P5119 245 5′ P4974 P5120 246 2 11-13 3′ P4976 P5121 247 5′ P4974 P5122 248 3 20-22 3′ P4976 P5123 249 5′ P4974 P5124 250 4 21-23 3′ P4976 P5125 251 5′ P4974 P5126 252 5 22-24 3′ P4976 P5127 253 5′ P4974 P5128 254 6 23-25 3′ P4976 P5129 255 5′ P4974 P5130 256 7 24-26 3′ P4976 P5131 257 5′ P4974 P5132 258 8 25-27 3′ P4976 P5133 259 5′ P4974 P5134 260 9 26-28 3′ P4976 P5135 261 5′ P4974 P5136 262 10 27-29 3′ P4976 P5137 263 5′ P4974 P5138 264 11 28-30 3′ P4976 P5139 265 5′ P4974 P5140 266 12 29-31 3′ P4976 P5141 267 5′ P4974 P5142 268 13 30-32 3′ P4976 P5143 269 5′ P4974 P5144 270 14 31-33 3′ P4976 P5145 271 5′ P4974 P5146 272 15 32-34 3′ P4976 P5147 273 5′ P4974 P5148 274 16 33-35 3′ P4976 P5149 275 5′ P4974 P5150 276 17 34-36 3′ P4976 P5151 277 5′ P4974 P5152 278 18 35-37 3′ P4976 P5153 279 5′ P4974 P5154 280 19 36-38 3′ P4976 P5155 281 5′ P4974 P5156 282 20 37-39 3′ P4976 P5157 283 5′ P4974 P5158 284 21 38-40 3′ P4976 P5159 285 5′ P4974 P5160 286 22 41-43 3′ P4976 P5161 287 5′ P4974 P5162 288 23 43-45 3′ P4976 P5163 289 5′ P4974 P5164 290 24 44-46 3′ P4976 P5165 291 5′ P4974 P5166 292 25 51-53 3′ P4976 P5167 293 5′ P4974 P5168 294 26 52-54 3′ P4976 P5169 295 5′ P4974 P5170 296 27 53-55 3′ P4976 P5171 297 5′ P4974 P5172 298 28 54-56 3′ P4976 P5173 299 5′ P4974 P5174 300 29 55-57 3′ P4976 P5175 301 5′ P4974 P5176 302 30 56-58 3′ P4976 P5177 303 5′ P4974 P5178 304 31 57-59 3′ P4976 P5179 305 5′ P4974 P5180 306 32 58-60 3′ P4976 P5181 307 5′ P4974 P5182 308 33 59-61 3′ P4976 P5183 309 5′ P4974 P5184 310 34 60-62 3′ P4976 P5185 311 5′ P4974 P5186 312 35 61-63 3′ P4976 P5187 313 5′ P4974 P5188 314 36 62-64 3′ P4976 P5189 315 5′ P4974 P5190 316 37 67-69 3′ P4976 P5191 317 5′ P4974 P5192 318 38 68-70 3′ P4976 P5193 319 5′ P4974 P5194 320 39 71-73 3′ P4976 P5195 321 5′ P4974 P5196 322 40 74-76 3′ P4976 P5197 323 5′ P4974 P5198 324 41 77-79 3′ P4976 P5199 325 5′ P4974 P5200 326 42 81-83 3′ P4976 P5201 327 5′ P4974 P5202 328 43 83-85 3′ P4976 P5203 329 5′ P4974 P5204 330 44 84-86 3′ P4976 P5205 331 5′ P4974 P5206 332 45 85-87 3′ P4976 P5207 333 5′ P4974 P5208 334 46 86-88 3′ P4976 P5209 335 5′ P4974 P5210 336 47 87-89 3′ P4976 P5211 337 5′ P4974 P5212 338 48 88-90 3′ P4976 P5213 339 5′ P4974 P5214 340 49 89-91 3′ P4976 P5215 341 5′ P4974 P5216 342 50 90-92 3′ P4976 P5217 343 5′ P4974 P5218 344 51 91-93 3′ P4976 P5219 345 5′ P4974 P5220 346 52 92-94 3′ P4976 P5221 347 5′ P4974 P5222 348 53 93-95 3′ P4976 P5223 349 5′ P4974 P5224 350 54 94-96 3′ P4976 P5225 351 5′ P4974 P5226 352 55 95-97 3′ P4976 P5227 353 5′ P4974 P5228 354 56 96-98 3′ P4976 P5229 355 5′ P4974 P5230 356 57 97-99 3′ P4976 P5231 357 5′ P4974 P5232 358 58  98-100 3′ P4976 P5233 359 5′ P4974 P5234 360 59  99-101 3′ P4976 P5235 361 5′ P4974 P5236 362 60 100-102 3′ P4976 P5237 363 5′ P4974 P5238 364 61 101-103 3′ P4976 P5239 365 5′ P4974 P5240 366 62 102-104 3′ P4976 P5241 367 5′ P4974 P5242 368 63 103-105 3′ P4976 P5243 369 5′ P4974 P5244 370 64 104-106 3′ P4976 P5245 371 5′ P4974 P5246 372 65 105-107 3′ P4976 P5247 373 5′ P4974 P5248 374 66 106-108 3′ P4976 P5249 375 5′ P4974 P5250 376 67 107-109 3′ P4976 P5251 377 5′ P4974 P5252 378 68 108-110 3′ P4976 P5253 379 5′ P4974 P5254 380 69 109-111 3′ P4976 P5255 381 5′ P4974 P5256 382 70 110-112 3′ P4976 P5257 383 5′ P4974 P5258 384 71 111-113 3′ P4976 P5259 385 5′ P4974 P5260 386 72 112-114 3′ P4976 P5261 387 5′ P4974 P5262 388 73 113-115 3′ P4976 P5263 389 5′ P4974 P5264 390 74 114-116 3′ P4976 P5265 391 5′ P4974 P5266 392 75 115-117 3′ P4976 P5267 393 5′ P4974 P5268 394 76 116-118 3′ P4976 P5269 395 5′ P4974 P5270 396 77 117-119 3′ P4976 P5271 397 5′ P4974 P5272 398 78 118-120 3′ P4976 P5273 399 5′ P4974 P5274 400 79 119-121 3′ P4976 P5275 401 5′ P4974 P5276 402 80 120-122 3′ P4976 P5277 403 5′ P4974 P5278 404 81 121-123 3′ P4976 P5279 405 5′ P4974 P5280 406 82 122-124 3′ P4976 P5281 407 5′ P4974 P5282 408 83 123-125 3′ P4976 P5283 409 5′ P4974 P5284 410 84 124-126 3′ P4976 P5285 411 5′ P4974 P5286 412 85 125-127 3′ P4976 P5287 413 5′ P4974 P5288 414 86 126-128 3′ P4976 P5289 415 5′ P4974 P5290 416 87 129-131 3′ P4976 P5291 417 5′ P4974 P5292 418 88 132-134 3′ P4976 P5293 419 5′ P4974 P5294 420 89 133-135 3′ P4976 P5295 421 5′ P4974 P5296 422 90 134-136 3′ P4976 P5297 423 5′ P4974 P5298 424 91 135-137 3′ P4976 P5299 425 5′ P4974 P5300 426 92 136-138 3′ P4976 P5301 427 5′ P4974 P5302 428 93 137-139 3′ P4976 P5303 429 5′ P4974 P5304 430 94 138-140 3′ P4976 P5305 431 5′ P4974 P5306 432 95 139-141 3′ P4976 P5307 433 5′ P4974 P5308 434 96 140-142 3′ P4976 P5309 435 5′ P4974 P5310 436 97 141-143 3′ P4976 P5311 437 5′ P4974 P5312 438 98 142-144 3′ P4976 P5313 439 5′ P4974 P5314 440 99 143-145 3′ P4976 P5315 441 5′ P4974 P5316 442 100 144-146 3′ P4976 P5317 443 5′ P4974 P5318 444 101 145-147 3′ P4976 P5319 445 5′ P4974 P5320 446 102 146-148 3′ P4976 P5321 447 5′ P4974 P5322 448 103 147-149 3′ P4976 P5323 449 5′ P4974 P5324 450 104 148-150 3′ P4976 P5325 451 5′ P4974 P5326 452 105 149-151 3′ P4976 P5327 453 5′ P4974 P5328 454 106 150-152 3′ P4976 P5329 455 5′ P4974 P5330 456 107 151-153 3′ P4976 P5331 457 5′ P4974 P5332 458 108 158-160 3′ P4976 P5333 459 5′ P4974 P5334 460 109 159-161 3′ P4976 P5335 461 5′ P4974 P5336 462 110 160-162 3′ P4976 P5337 463 5′ P4974 P5338 464 111 163-165 3′ P4976 P5339 465 5′ P4974 P5340 466 112 164-166 3′ P4976 P5341 467 5′ P4974 P5342 468 113 167-169 3′ P4976 P5343 469 5′ P4974 P5344 470 114 168-170 3′ P4976 P5345 471 5′ P4974 P5346 472 115 169-171 3′ P4976 P5347 473 5′ P4974 P5348 474 116 170-172 3′ P4976 P5349 475 5′ P4974 P5350 476 117 171-173 3′ P4976 P5351 477 5′ P4974 P5352 478 118 172-174 3′ P4976 P5353 479 5′ P4974 P5354 480 119 182-184 3′ P4976 P5355 481 5′ P4974 P5356 482 120 183-185 3′ P4976 P5357 483 5′ P4974 P5358 484 121 184-186 3′ P4976 P5359 485 5′ P4974 P5360 486 122 185-187 3′ P4976 P5361 487 5′ P4974 P5362 488 123 186-188 3′ P4976 P5363 489 5′ P4974 P5364 490 124 192-194 3′ P4976 P5365 491 5′ P4974 P5366 492 125 194-196 3′ P4976 P5367 493 5′ P4974 P5368 494 126 195-197 3′ P4976 P5369 495 5′ P4974 P5370 496 127 196-198 3′ P4976 P5371 497 5′ P4974 P5372 498 128 197-199 3′ P4976 P5373 499 5′ P4974 P5374 500 129 198-200 3′ P4976 P5375 501 5′ P4974 P5376 502 130 203-205 3′ P4976 P5377 503 5′ P4974 P5378 504 131 210-212 3′ P4976 P5379 505 5′ P4974 P5380 506 132 211-213 3′ P4976 P5381 507 5′ P4974 P5382 508 133 216-218 3′ P4976 P5383 509 5′ P4974 P5384 510 134 217-219 3′ P4976 P5385 511 5′ P4974 P5386 512 135 218-220 3′ P4976 P5387 513 5′ P4974 P5388 514 136 219-221 3′ P4976 P5389 515 5′ P4974 P5390 516 137 230-232 3′ P4976 P5391 517 5′ P4974 P5392 518 138 231-233 3′ P4976 P5393 519 5′ P4974 P5394 520 139 232-234 3′ P4976 P5395 521 5′ P4974 P5396 522 140 238-240 3′ P4976 P5397 523 5′ P4974 P5398 524 141 240-242 3′ P4976 P5399 525 5′ P4974 P5400 526 142 246-248 3′ P4976 P5401 527 5′ P4974 P5402 528 143 255-257 3′ P4976 P5403 529 5′ P4974 P5404 530 144 258-260 3′ P4976 P5405 531 5′ P4974 P5406 532 145 265-267 3′ P4976 P5407 533 5′ P4974 P5408 534

RCL5 Variants

“RCL5” refers to a set of combinatorial variants created by PCR fusion using several BPN′ mutants as parent (template) molecules. The mutations introduced in each parent plasmid are shown in Table 11-7 and the mutagenic primers used to create the mutants are indicated in Table 11-8.

TABLE 11-7 List of Parent Plasmids and Mutations Introduced in the RCL5 Variants Combi- natorial Mutations Variant # Parent Plasmids Introduced 1 G97A-G128A-Y217Q-T22N-S24A N61P-N62S 2 G97A-G128A-Y217Q-T22N-S24A T55P 3 G97A-G128A-Y217Q-T22N-S24A N61P-S63H 4 G97A-G128A-Y217Q-T22N-S24A Q59S-N61P 5 G97A-G128A-Y217Q-T22N-S24A L75S-N76Y 6 G97A-G128A-Y217Q-T22N-S24A P86S-S87G-A88V 7 G97A-G128A-Y217Q-T22N-S24A S87G-A88V-S89A 8 G97A-G128A-Y217Q-T22N-S24A S87T-A88L-S89G 9 G97A-G128A-Y217Q-T22N-S24A P129Q-S130G-G131S 10 G97A-G128A-Y217Q-T22N-S24A V203Y 11 G97A-G128A-Y217Q-T22N-S24A G211R-N212S-K213V 12 G97A-G128A-Y217Q-S24G-N25G N61P-N62S 13 G97A-G128A-Y217Q-S24G-N25G T55P 14 G97A-G128A-Y217Q-S24G-N25G N61P-S63H 15 G97A-G128A-Y217Q-S24G-N25G Q59S-N61P 16 G97A-G128A-Y217Q-S24G-N25G L75S-N76Y 17 G97A-G128A-Y217Q-S24G-N25G P86S-S87G-A88V 18 G97A-G128A-Y217Q-S24G-N25G S87G-A88V-S89A 19 G97A-G128A-Y217Q-S24G-N25G S87T-A88L-S89G 20 G97A-G128A-Y217Q-S24G-N25G P129Q-S130G-G131S 21 G97A-G128A-Y217Q-S24G-N25G V203Y 22 G97A-G128A-Y217Q-S24G-N25G G211R-N212S-K213V 23 G97A-G128A-Y217Q-S24R N61P-N62S 24 G97A-G128A-Y217Q-S24R T55P 25 G97A-G128A-Y217Q-S24R N61P-S63H 26 G97A-G128A-Y217Q-S24R Q59S-N61P 27 G97A-G128A-Y217Q-S24R L75S-N76Y 28 G97A-G128A-Y217Q-S24R P86S-S87G-A88V 29 G97A-G128A-Y217Q-S24R S87G-A88V-S89A 30 G97A-G128A-Y217Q-S24R S87T-A88L-S89G 31 G97A-G128A-Y217Q-S24R P129Q-S130G-G131S 32 G97A-G128A-Y217Q-S24R V203Y 33 G97A-G128A-Y217Q-S24R G211R-N212S-K213V 34 G97A-G128A-Y217Q-G23A-S24G-N25G N61P-N62S 35 G97A-G128A-Y217Q-G23A-S24G-N25G T55P 36 G97A-G128A-Y217Q-G23A-S24G-N25G N61P-S63H 37 G97A-G128A-Y217Q-G23A-S24G-N25G Q59S-N61P 38 G97A-G128A-Y217Q-G23A-S24G-N25G L75S-N76Y 39 G97A-G128A-Y217Q-G23A-S24G-N25G P86S-S87G-A88V 40 G97A-G128A-Y217Q-G23A-S24G-N25G S87G-A88V-S89A 41 G97A-G128A-Y217Q-G23A-S24G-N25G S87T-A88L-S89G 42 G97A-G128A-Y217Q-G23A-S24G-N25G P129Q-S130G-G131S 43 G97A-G128A-Y217Q-G23A-S24G-N25G V203Y 44 G97A-G128A-Y217Q-G23A-S24G-N25G G211R-N212S-K213V 45 G97A-G128A-Y217Q-N61P-N62S L75S-N76Y 46 G97A-G128A-Y217Q-N61P-N62S P86S-S87G-A88V 47 G97A-G128A-Y217Q-N61P-N62S S87G-A88V-S89A 48 G97A-G128A-Y217Q-N61P-N62S S87T-A88L-S89G 49 G97A-G128A-Y217Q-N61P-N62S P129Q-S130G-G131S 50 G97A-G128A-Y217Q-N61P-N62S V203Y 51 G97A-G128A-Y217Q-N61P-N62S G211R-N212S-K213V 52 G97A-G128A-Y217Q-T55P L75S-N76Y 53 G97A-G128A-Y217Q-T55P P86S-S87G-A88V 54 G97A-G128A-Y217Q-T55P S87G-A88V-S89A 55 G97A-G128A-Y217Q-T55P S87T-A88L-S89G 56 G97A-G128A-Y217Q-T55P P129Q-S130G-G131S 57 G97A-G128A-Y217Q-T55P V203Y 58 G97A-G128A-Y217Q-T55P G211R-N212S-K213V 59 G97A-G128A-Y217Q-N61P-S63H L75S-N76Y 60 G97A-G128A-Y217Q-N61P-S63H P86S-S87G-A88V 61 G97A-G128A-Y217Q-N61P-S63H S87G-A88V-S89A 62 G97A-G128A-Y217Q-N61P-S63H S87T-A88L-S89G 63 G97A-G128A-Y217Q-N61P-S63H P129Q-S130G-G131S 64 G97A-G128A-Y217Q-N61P-S63H V203Y 65 G97A-G128A-Y217Q-N61P-S63H G211R-N212S-K213V 66 G97A-G128A-Y217Q-Q59S-N61P L75S-N76Y 67 G97A-G128A-Y217Q-Q59S-N61P P86S-S87G-A88V 68 G97A-G128A-Y217Q-Q59S-N61P S87G-A88V-S89A 69 G97A-G128A-Y217Q-Q59S-N61P S87T-A88L-S89G 70 G97A-G128A-Y217Q-Q59S-N61P P129Q-S130G-G131S 71 G97A-G128A-Y217Q-Q59S-N61P V203Y 72 G97A-G128A-Y217Q-Q59S-N61P G211R-N212S-K213V 73 G97A-G128A-Y217Q-L75S-N76Y P86S-S87G-A88V 74 G97A-G128A-Y217Q-L75S-N76Y S87G-A88V-S89A 75 G97A-G128A-Y217Q-L75S-N76Y S87T-A88L-S89G 76 G97A-G128A-Y217Q-L75S-N76Y P129Q-S130G-G131S 77 G97A-G128A-Y217Q-L75S-N76Y V203Y 78 G97A-G128A-Y217Q-L75S-N76Y G211R-N212S-K213V 79 G97A-G128A-Y217Q-P86S-S87G-A88V P129Q-S130G-G131S 80 G97A-G128A-Y217Q-P86S-S87G-A88V V203Y 81 G97A-G128A-Y217Q-P86S-S87G-A88V G211R-N212S-K213V 82 G97A-G128A-Y217Q-S87G-A88V-S89A P129Q-S130G-G131S 83 G97A-G128A-Y217Q-S87G-A88V-S89A V203Y 84 G97A-G128A-Y217Q-S87G-A88V-S89A G211R-N212S-K213V 85 G97A-G128A-Y217Q-S87T-A88L-S89G P129Q-S130G-G131S 86 G97A-G128A-Y217Q-S87T-A88L-S89G V203Y 87 G97A-G128A-Y217Q-S87T-A88L-S89G G211R-N212S-K213V 88 G97A-G128A-Y217Q-P129Q-S130G-G131S V203Y 89 G97A-G128A-Y217Q-P129Q-S130G-G131S G211R-N212S-K213V 90 G97A-G128A-Y217Q-V203Y G211R-N212S-K213V

TABLE 11-8 Primers Used to Create RCL5 Combinatorial Variants Common 3′ & 5′ Gene Mutations Gene Flanking Primer SEQ Introduced Fragments Primer Names Name ID NO: T22N-S24A 3′ P4976 P5432 535 5′ P4974 P5433 536 S24G-N25G 3′ P4976 P5434 537 5′ P4974 P5435 538 S24R 3′ P4976 P5436 539 5′ P4974 P5437 540 G23A-S24G-N25G 3′ P4976 P5438 541 5′ P4974 P5439 542 N61P-N62S 3′ P4976 P5440 543 5′ P4974 P5441 544 T55P 3′ P4976 P5442 545 5′ P4974 P5443 546 N61P-S63H 3′ P4976 P5444 547 5′ P4974 P5445 548 Q59S-N61P 3′ P4976 P5446 549 5′ P4974 P5447 550 L75S-N76Y 3′ P4976 P5448 551 5′ P4974 P5449 552 P86S-S87G-A88V 3′ P4976 P5450 553 5′ P4974 P5451 554 S87G-A88V-S89A 3′ P4976 P5452 555 5′ P4974 P5453 556 S87T-A88L-S89G 3′ P4976 P5454 557 5′ P4974 P5455 558 P129Q-S130G-G131S 3′ P4976 P5456 559 5′ P4974 P5457 560 V203Y 3′ P4976 P5458 561 5′ P4974 P5459 562 G211R-N212S-K213V 3′ P4976 P5460 563 5′ P4974 P5461 564

To create each mutant, two PCR reactions were carried out using either the common 3′ gene-flanking primer (P4976, CCTCTCGGTTATGAGTTAGTTC; SEQ ID NO:61) and the mutagenic primer, or the common 5′gene-flanking primer (P4974, GCCTCACATTTGTGCCACCTA; SEQ ID NO:60) and mutagenic primer as shown for each library in Table 11-8. These PCR reactions generated two PCR fragments, one encoding the 5′ half of the mutant BPN′ gene (5′ gene fragment) and the other encoding the 3′ half of the mutant BPN′ gene (3′ gene fragment). Each PCR amplification reaction contained 30 pmol of each primer and 100 ng of the parent molecules listed in Table 11-7. Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 40 sec. Following amplification, the 5′ and 3′ gene fragments were gel-purified by the QIAGEN® gel-band purification kit, mixed (50 ng of each fragment) and amplified by PCR once again using the primers P4973 (SEQ ID NO:62) and P4950 (SEQ ID NO:63) to generate the full-length gene fragment. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min. The full-length DNA fragment was gel-purified by the QIAGEN® gel-band purification kit, digested by the BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ partial opt that also was digested with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification by Illustra Templiphi kit according to the manufacturer's instructions (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, the colonies with halos were inoculated in 120 μl of LB media containing 10 μg/mL neomycin.

RCL 6 Combinatorial Libraries

“RCL6” refers to a group of combinatorial libraries created by PCR fusion using several BPN′ mutants as parent (template) molecules. A mixture of BPN′ mutants were used as templates (parent molecules) in the construction of each of these libraries. The five different mixes of parent molecules used to create these libraries are provided in Table 11-9, and the mutations introduced in each library are listed in Table 11-10.

To create each mutant, two PCR reactions were carried out using either the common 3′ gene-flanking primer (P4976, SEQ ID NO:61) and the mutagenic primer, or the common 5′gene-flanking primer (P4974, SEQ ID NO:60) and mutagenic primer as shown for each library in Table 11-10. These PCR reactions generated two PCR fragments, one encoding the 5′ half of the mutant BPN′ gene (5′ gene fragment) and the other encoding the 3′ half of the mutant BPN′ gene (3′ gene fragment). Each PCR amplification reaction contained 30 pmol of each primer and 100 ng of the parent molecules listed in Table 11-9 Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 40 sec. Following amplification, the 5′ and 3′ gene fragments were gel-purified by the QIAGEN® gel-band purification kit, mixed (50 ng of each fragment) and amplified by PCR once again using the primers P4973 (SEQ ID NO:62) and P4950 (SEQ ID NO:63) to generate the full-length gene fragment. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min. The full-length DNA fragment was gel-purified by the QIAGEN® gel-band purification kit, digested using BamHI and HindIII restriction enzymes and ligated with the pHPLT-BPN′ partial opt vector that also was digested with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification by Illustra Templiphi kit according to the manufacturer's instructions (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, the colonies with halos were inoculated in 120 μl of LB media containing 10 μg/mL neomycin.

TABLE 11-9 Parent Molecules of BPN′ Used to Create RCL6 Libraries Mixes of Parent Molecules Mix 1 Mix 2 Mix 3 Mix 4 Mix 5 G97A-G128A- G97A-G128A- G97A-G128A- G97A-G128A- G97A-G128A- Y217Q-S24G- Y217Q-T55P Y217Q-L75S- Y217Q-P86S- Y217Q-P129Q- N25G N76Y S87G-A88V S130G-G131S G97A-G128A- G97A-G128A- G97A-G128A- Y217Q-S24R Y217Q-N61P- Y217Q-S87G- S63H A88V-S89A G97A-G128A- G97A-G128A- G97A-G128A- Y217Q-G23A- Y217Q-Q59S- Y217Q-S87T- S24G-N25G N61P A88L-S89G

TABLE 11-10 Mutations Introduced in the RCL6 Libraries Common 5′ & 3′ Gene Flanking Mutagenic SEQ Gene Primer Primer ID Mutations Introduced Fragment Names Name NO: V68C, A69G 3′ P4976 P5462 565 5′ P4974 P5463 566 V72I, A73G, delA74, L75S 3′ P4976 P5464 567 5′ P4974 P5465 568 L75H, N76G 3′ P4976 P5466 569 5′ P4974 P5467 570 L75R, N76G, N77S 3′ P4976 P5468 571 5′ P4974 P5469 572 L75G, N76G, N77G 3′ P4976 P5470 573 5′ P4974 P5471 574 A92G 3′ P4976 P5472 575 5′ P4974 P5473 576 delV93, K94S, V95C, 3′ P4976 P5474 577 L96S 5′ P4974 P5475 578 V121I_I122S_N123C 3′ P4976 P5476 579 5′ P4974 P5477 580 V121L_N123C 3′ P4976 P5478 581 5′ P4974 P5479 582 I122C_N123S_M124L 3′ P4976 P5480 583 5′ P4974 P5481 584 N123C 3′ P4976 P5482 585 5′ P4974 P5483 586 M124I 3′ P4976 P5484 587 5′ P4974 P5485 588 M124V 3′ P4976 P5486 589 5′ P4974 P5487 590 M124V-L126A 3′ P4976 P5488 591 5′ P4974 P5489 592 L126F, delP129 3′ P4976 P5490 593 5′ P4974 P5491 594 G127Y 3′ P4976 P5492 595 5′ P4974 P5493 596 G127S_P129D 3′ P4976 P5494 597 5′ P4974 P5495 598 G127N, P129R 3′ P4976 P5496 599 5′ P4974 P5497 600 G128N, insS, P129S 3′ P4976 P5498 601 5′ P4974 P5499 602 G128S_P129V 3′ P4976 P5500 603 5′ P4974 P5501 604 G128S, P129D 3′ P4976 P5502 605 5′ P4974 P5503 606 G128S, P129G 3′ P4976 P5504 607 5′ P4974 P5505 608 H128H, P129Y 3′ P4976 P5506 609 5′ P4974 P5507 610 P129D 3′ P4976 P5508 611 5′ P4974 P5509 612 P129E 3′ P4976 P5510 613 5′ P4974 P5511 614 P129V 3′ P4976 P5512 615 5′ P4974 P5513 616 P129G, delS130 3′ P4976 P5514 617 5′ P4974 P5515 618 P129H, delS130, S132N 3′ P4976 P5516 619 5′ P4974 P5517 620 A134T 3′ P4976 P5518 621 5′ P4974 P5519 622 G97R, insG, A98C 3′ P4976 P5520 623 5′ P4974 P5521 624 A98G, D99G 3′ P4976 P5522 625 5′ P4974 P5523 626 A98G, insR 3′ P4976 P5524 627 5′ P4974 P5525 628 A98D, D99G 3′ P4976 P5526 629 5′ P4974 P5527 630 A98H, D99G, G100D 3′ P4976 P5528 631 5′ P4974 P5529 632 D99R, insN 3′ P4976 P5530 633 5′ P4974 P5531 634 D99V, S101D 3′ P4976 P5532 635 5′ P4974 P5533 636 D99C, insS 3′ P4976 P5534 637 5′ P4974 P5535 638 G100S 3′ P4976 P5536 639 5′ P4974 P5537 640 G100S, S101V 3′ P4976 P5538 641 5′ P4974 P5539 642 G100D 3′ P4976 P5540 643 5′ P4974 P5541 644 G100N 3′ P4976 P5542 645 5′ P4974 P5543 646 S100N, S101L 3′ P4976 P5544 647 5′ P4974 P5545 648 S101G 3′ P4976 P5546 649 5′ P4974 P5547 650 S101D 3′ P4976 P5548 651 5′ P4974 P5549 652 S101V, Q103N 3′ P4976 P5550 653 5′ P4974 P5551 654 S101E 3′ P4976 P5552 655 5′ P4974 P5553 656 A116S, N117G, N118R 3′ P4976 P5554 657 5′ P4974 P5555 658 A116G, N117R 3′ P4976 P5556 659 5′ P4974 P5557 660 A116N, N117S, N118G 3′ P4976 P5558 661 5′ P4974 P5559 662 M222Q 3′ P4976 P5560 663 5′ P4974 P5561 664 S24R 3′ P4976 P5562 665 5′ P4974 P5563 666 N25Y 3′ P4976 P5564 667 5′ P4974 P5565 668 P52D 3′ P4976 P5566 669 5′ P4974 P5567 670 S63T 3′ P4976 P5568 671 5′ P4974 P5569 672 N61E 3′ P4976 P5570 673 5′ P4974 P5571 674 N61P 3′ P4976 P5572 675 5′ P4974 P5573 676 N62Q 3′ P4976 P5574 677 5′ P4974 P5575 678 N62D 3′ P4976 P5576 679 5′ P4974 P5577 680 S63Q 3′ P4976 P5578 681 5′ P4974 P5579 682 V68A 3′ P4976 P5580 683 5′ P4974 P5581 684 S87D 3′ P4976 P5582 685 5′ P4974 P5583 686 L96T 3′ P4976 P5584 687 5′ P4974 P5585 688 L126A 3′ P4976 P5586 689 5′ P4974 P5587 690 L126T 3′ P4976 P5588 691 5′ P4974 P5589 692 S125A 3′ P4976 P5590 693 5′ P4974 P5591 694 S130P 3′ P4976 P5592 695 5′ P4974 P5593 696 P129L 3′ P4976 P5594 697 5′ P4974 P5595 698 P129E 3′ P4976 P5596 699 5′ P4974 P5597 700 P129S 3′ P4976 P5598 701 5′ P4974 P5599 702 P40E 3′ P4976 P5600 703 5′ P4974 P5601 704 Y6Q 3′ P4976 P5602 705 5′ P4974 P5603 706 G102A 3′ P4976 P5604 707 5′ P4974 P5605 708 S101N 3′ P4976 P5606 709 5′ P4974 P5607 710 G100E 3′ P4976 P5608 711 5′ P4974 P5609 712 I115V 3′ P4976 P5610 713 5′ P4974 P5611 714 A144K 3′ P4976 P5612 715 5′ P4974 P5613 716 S145D 3′ P4976 P5614 717 5′ P4974 P5615 718 S159K 3′ P4976 P5616 719 5′ P4974 P5617 720 S162K 3′ P4976 P5618 721 5′ P4974 P5619 722 V147P 3′ P4976 P5620 723 5′ P4974 P5621 724 S161P 3′ P4976 P5622 725 5′ P4974 P5623 726 A187D 3′ P4976 P5624 727 5′ P4974 P5625 728 F189D 3′ P4976 P5626 729 5′ P4974 P5627 730 L267V 3′ P4976 P5628 731 5′ P4974 P5629 732 Q206E 3′ P4976 P5630 733 5′ P4974 P5631 734 K213T 3′ P4976 P5632 735 5′ P4974 P5633 736 K213L 3′ P4976 P5634 737 5′ P4974 P5635 738 K265N 3′ P4976 P5636 739 5′ P4974 P5637 740 N240K 3′ P4976 P5638 741 5′ P4974 P5639 742 P239R 3′ P4976 P5640 743 5′ P4974 P5641 744 T242R 3′ P4976 P5642 745 5′ P4974 P5643 746 S89Y 3′ P4976 P5644 747 5′ P4974 P5645 748 P129Q 3′ P4976 P5646 749 5′ P4974 P5647 750 G211T 3′ P4976 P5648 751 5′ P4974 P5649 752 I111V 3′ P4976 P5650 753 5′ P4974 P5651 754

RCL 7 Combinatorial Variants

“RCL7” refers to a set of combinatorial variants created by PCR fusion using several BPN′ mutants as parent (template) plasmid. The mutations introduced in each parent plasmid are listed in Table 11-11, and the mutagenic primers used to create the mutants are described in Table 11-10.

To create each mutant, two PCR reactions were carried out using either the common 3′ gene-flanking primer (P4976, SEQ ID NO:61) and the mutagenic primer, or the common 5′gene-flanking primer (P4974, SEQ ID NO:60) and mutagenic primer as shown for each library in Table 11-10. These PCR reactions generated two PCR fragments, one encoding the 5′ half of the mutant BPN′ gene (5′ gene fragment) and the other encoding the 3′ half of the mutant BPN′ gene (3′ gene fragment). Each PCR amplification reaction contained 30 pmol of each primer and 100 ng of the parent molecules listed in Table 11-11. Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 40 sec. Following amplification, the 5′ and 3′ gene fragments were gel-purified using a QIAGEN® gel-band purification kit, mixed (50 ng of each fragment) and amplified by PCR once again using the primers P4973 (SEQ ID NO:62) and P4950 (SEQ ID NO:63) to generate the full-length gene fragment. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min. The full-length DNA fragment was gel-purified using a QIAGEN® gel-band purification kit, digested using BamHI and HindIII restriction enzymes, and ligated with the pHPLT-BPN′ partial opt that also was digested with the same restriction enzymes. Ligation mixtures were amplified using rolling circle amplification by Illustra Templiphi kit according to the manufacturer's instructions (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, the colonies with halos were inoculated in 120 μl of LB media containing 10 μg/mL neomycin.

TABLE 11-11 List of Parent Plasmids and Introduced Mutations in the RCL7 variants Combi- natorial Mutation(s) Variant # Parent Plasmid Introduced 1 G97A-G128A-Y217Q-T55P S24R 2 G97A-G128A-Y217Q-N61E S24R 3 G97A-G128A-Y217Q-N61P-S63H S24R 4 G97A-G128A-Y217Q-L75S-N76Y S24R 5 G97A-G128A-Y217Q-S87T-A88L- S24R S89G 6 G97A-G128A-Y217Q-S89Y S24R 7 G97A-G128A-Y217Q-I111V S24R 8 G97A-G128A-Y217Q-I115V S24R 9 G97A-G128A-Y217Q-P129Q-S130G- S24R G131S 10 G97A-G128A-Y217Q-P129Q S24R 11 G97A-G128A-Y217Q-A134T S24R 12 G97A-G128A-Y217Q-A144K S24R 13 G97A-G128A-Y217Q-S145D S24R 14 G97A-G128A-Y217Q-S159K S24R 15 G97A-G128A-Y217Q-S162K S24R 16 G97A-G128A-Y217Q-S161P-S78N S24R 17 G97A-G128A-Y217Q-V203Y S24R 18 G97A-G128A-Y217Q-G211T-S78N S24R 19 G97A-G128A-Y217Q-K213T S24R 20 G97A-G128A-Y217Q-P239R S24R 21 G97A-G128A-Y217Q-N240K S24R 22 G97A-G128A-Y217Q-L267V-S78N S24R 23 G97A-G128A-Y217Q-A273S-S78N S24R 24 G97A-G128A-Y217Q-L75S-N76Y T55P 25 G97A-G128A-Y217Q-S87T-A88L- T55P S89G 26 G97A-G128A-Y217Q-S89Y T55P 27 G97A-G128A-Y217Q-I111V T55P 28 G97A-G128A-Y217Q-I115V T55P 29 G97A-G128A-Y217Q-P129Q-S130G- T55P G131S 30 G97A-G128A-Y217Q-P129Q T55P 31 G97A-G128A-Y217Q-A134T T55P 32 G97A-G128A-Y217Q-A144K T55P 33 G97A-G128A-Y217Q-S145D T55P 34 G97A-G128A-Y217Q-S159K T55P 35 G97A-G128A-Y217Q-S162K T55P 36 G97A-G128A-Y217Q-S161P T55P 37 G97A-G128A-Y217Q-V203Y T55P 38 G97A-G128A-Y217Q-G211T T55P 39 G97A-G128A-Y217Q-K213T T55P 40 G97A-G128A-Y217Q-P239R T55P 41 G97A-G128A-Y217Q-N240K T55P 42 G97A-G128A-Y217Q-L267V T55P 43 G97A-G128A-Y217Q-A273S T55P 44 G97A-G128A-Y217Q-L75S-N76Y N61E 45 G97A-G128A-Y217Q-S87T-A88L- N61E S89G 46 G97A-G128A-Y217Q-S89Y N61E 47 G97A-G128A-Y217Q-I111V N61E 48 G97A-G128A-Y217Q-I115V N61E 49 G97A-G128A-Y217Q-P129Q-S130G- N61E G131S 50 G97A-G128A-Y217Q-P129Q N61E 51 G97A-G128A-Y217Q-A134T N61E 52 G97A-G128A-Y217Q-A144K N61E 53 G97A-G128A-Y217Q-S145D N61E 54 G97A-G128A-Y217Q-S159K N61E 55 G97A-G128A-Y217Q-S162K N61E 56 G97A-G128A-Y217Q-S161P N61E 57 G97A-G128A-Y217Q-V203Y N61E 58 G97A-G128A-Y217Q-G211T N61E 59 G97A-G128A-Y217Q-K213T N61E 60 G97A-G128A-Y217Q-P239R N61E 61 G97A-G128A-Y217Q-N240K N61E 62 G97A-G128A-Y217Q-L267V N61E 63 G97A-G128A-Y217Q-A273S N61E 64 G97A-G128A-Y217Q-L75S-N76Y N61P-S63H 65 G97A-G128A-Y217Q-S87T-A88L- N61P-S63H S89G 66 G97A-G128A-Y217Q-S89Y N61P-S63H 67 G97A-G128A-Y217Q-I111V N61P-S63H 68 G97A-G128A-Y217Q-I115V N61P-S63H 69 G97A-G128A-Y217Q-P129Q-S130G- N61P-S63H G131S 70 G97A-G128A-Y217Q-P129Q N61P-S63H 71 G97A-G128A-Y217Q-A134T N61P-S63H 72 G97A-G128A-Y217Q-A144K N61P-S63H 73 G97A-G128A-Y217Q-S145D N61P-S63H 74 G97A-G128A-Y217Q-S159K N61P-S63H 75 G97A-G128A-Y217Q-S162K N61P-S63H 76 G97A-G128A-Y217Q-S161P N61P-S63H 77 G97A-G128A-Y217Q-V203Y N61P-S63H 78 G97A-G128A-Y217Q-G211T N61P-S63H 79 G97A-G128A-Y217Q-K213T N61P-S63H 80 G97A-G128A-Y217Q-P239R N61P-S63H 81 G97A-G128A-Y217Q-N240K N61P-S63H 82 G97A-G128A-Y217Q-L267V N61P-S63H 83 G97A-G128A-Y217Q-A273S N61P-S63H 84 G97A-G128A-Y217Q-S87T-A88L- L75S-N76Y S89G 85 G97A-G128A-Y217Q-S89Y L75S-N76Y 86 G97A-G128A-Y217Q-I111V L75S-N76Y 87 G97A-G128A-Y217Q-I115V L75S-N76Y 88 G97A-G128A-Y217Q-P129Q-S130G- L75S-N76Y G131S 89 G97A-G128A-Y217Q-P129Q L75S-N76Y 90 G97A-G128A-Y217Q-A134T L75S-N76Y 91 G97A-G128A-Y217Q-A144K L75S-N76Y 92 G97A-G128A-Y217Q-S145D L75S-N76Y 93 G97A-G128A-Y217Q-S159K L75S-N76Y 94 G97A-G128A-Y217Q-S162K L75S-N76Y 95 G97A-G128A-Y217Q-S161P L75S-N76Y 96 G97A-G128A-Y217Q-V203Y L75S-N76Y 97 G97A-G128A-Y217Q-G211T L75S-N76Y 98 G97A-G128A-Y217Q-K213T L75S-N76Y 99 G97A-G128A-Y217Q-P239R L75S-N76Y 100 G97A-G128A-Y217Q-N240K L75S-N76Y 101 G97A-G128A-Y217Q-L267V L75S-N76Y 102 G97A-G128A-Y217Q-A273S L75S-N76Y 103 G97A-G128A-Y217Q-I111V S87T-A88L-S89G 104 G97A-G128A-Y217Q-I115V S87T-A88L-S89G 105 G97A-G128A-Y217Q-P129Q-S130G- S87T-A88L-S89G G131S 106 G97A-G128A-Y217Q-P129Q S87T-A88L-S89G 107 G97A-G128A-Y217Q-A134T S87T-A88L-S89G 108 G97A-G128A-Y217Q-A144K S87T-A88L-S89G 109 G97A-G128A-Y217Q-S145D S87T-A88L-S89G 110 G97A-G128A-Y217Q-S159K S87T-A88L-S89G 111 G97A-G128A-Y217Q-S162K S87T-A88L-S89G 112 G97A-G128A-Y217Q-S161P S87T-A88L-S89G 113 G97A-G128A-Y217Q-V203Y S87T-A88L-S89G 114 G97A-G128A-Y217Q-G211T S87T-A88L-S89G 115 G97A-G128A-Y217Q-K213T S87T-A88L-S89G 116 G97A-G128A-Y217Q-P239R S87T-A88L-S89G 117 G97A-G128A-Y217Q-N240K S87T-A88L-S89G 118 G97A-G128A-Y217Q-L267V S87T-A88L-S89G 119 G97A-G128A-Y217Q-A273S S87T-A88L-S89G 120 G97A-G128A-Y217Q-I111V S89Y 121 G97A-G128A-Y217Q-I115V S89Y 122 G97A-G128A-Y217Q-P129Q-S130G- S89Y G131S 123 G97A-G128A-Y217Q-P129Q S89Y 124 G97A-G128A-Y217Q-A134T S89Y 125 G97A-G128A-Y217Q-A144K S89Y 126 G97A-G128A-Y217Q-S145D S89Y 127 G97A-G128A-Y217Q-S159K S89Y 128 G97A-G128A-Y217Q-S162K S89Y 129 G97A-G128A-Y217Q-S161P S89Y 130 G97A-G128A-Y217Q-V203Y S89Y 131 G97A-G128A-Y217Q-G211T S89Y 132 G97A-G128A-Y217Q-K213T S89Y 133 G97A-G128A-Y217Q-P239R S89Y 134 G97A-G128A-Y217Q-N240K S89Y 135 G97A-G128A-Y217Q-L267V S89Y 136 G97A-G128A-Y217Q-A273S S89Y 137 G97A-G128A-Y217Q-P129Q-S130G- I111V G131S 138 G97A-G128A-Y217Q-P129Q I111V 139 G97A-G128A-Y217Q-A134T I111V 140 G97A-G128A-Y217Q-A144K I111V 141 G97A-G128A-Y217Q-S145D I111V 142 G97A-G128A-Y217Q-S159K I111V 143 G97A-G128A-Y217Q-S162K I111V 144 G97A-G128A-Y217Q-S161P I111V 145 G97A-G128A-Y217Q-V203Y I111V 146 G97A-G128A-Y217Q-G211T I111V 147 G97A-G128A-Y217Q-K213T I111V 148 G97A-G128A-Y217Q-P239R I111V 149 G97A-G128A-Y217Q-N240K I111V 150 G97A-G128A-Y217Q-L267V I111V 151 G97A-G128A-Y217Q-A273S I111V 152 G97A-G128A-Y217Q-P129Q-S130G- I115V G131S 153 G97A-G128A-Y217Q-P129Q I115V 154 G97A-G128A-Y217Q-A134T I115V 155 G97A-G128A-Y217Q-A144K I115V 156 G97A-G128A-Y217Q-S145D I115V 157 G97A-G128A-Y217Q-S159K I115V 158 G97A-G128A-Y217Q-S162K I115V 159 G97A-G128A-Y217Q-S161P I115V 160 G97A-G128A-Y217Q-V203Y I115V 161 G97A-G128A-Y217Q-G211T I115V 162 G97A-G128A-Y217Q-K213T I115V 163 G97A-G128A-Y217Q-P239R I115V 164 G97A-G128A-Y217Q-N240K I115V 165 G97A-G128A-Y217Q-L267V I115V 166 G97A-G128A-Y217Q-A273S I115V 167 G97A-G128A-Y217Q-A144K P129Q-S130G- G131S 168 G97A-G128A-Y217Q-S145D P129Q-S130G- G131S 169 G97A-G128A-Y217Q-S159K P129Q-S130G- G131S 170 G97A-G128A-Y217Q-S162K P129Q-S130G- G131S 171 G97A-G128A-Y217Q-S161P P129Q-S130G- G131S 172 G97A-G128A-Y217Q-V203Y P129Q-S130G- G131S 173 G97A-G128A-Y217Q-G211T P129Q-S130G- G131S 174 G97A-G128A-Y217Q-K213T P129Q-S130G- G131S 175 G97A-G128A-Y217Q-P239R P129Q-S130G- G131S 176 G97A-G128A-Y217Q-N240K P129Q-S130G- G131S 177 G97A-G128A-Y217Q-L267V P129Q-S130G- G131S 178 G97A-G128A-Y217Q-A273S P129Q-S130G- G131S 179 G97A-G128A-Y217Q-A144K P129Q 180 G97A-G128A-Y217Q-S145D P129Q 181 G97A-G128A-Y217Q-S159K P129Q 182 G97A-G128A-Y217Q-S162K P129Q 183 G97A-G128A-Y217Q-S161P P129Q 184 G97A-G128A-Y217Q-V203Y P129Q 185 G97A-G128A-Y217Q-G211T P129Q 186 G97A-G128A-Y217Q-K213T P129Q 187 G97A-G128A-Y217Q-P239R P129Q 188 G97A-G128A-Y217Q-N240K P129Q 189 G97A-G128A-Y217Q-L267V P129Q 190 G97A-G128A-Y217Q-A273S P129Q 191 G97A-G128A-Y217Q-A144K A134T 192 G97A-G128A-Y217Q-S145D A134T 193 G97A-G128A-Y217Q-S159K A134T 194 G97A-G128A-Y217Q-S162K A134T 195 G97A-G128A-Y217Q-S161P A134T 196 G97A-G128A-Y217Q-V203Y A134T 197 G97A-G128A-Y217Q-G211T A134T 198 G97A-G128A-Y217Q-K213T A134T 199 G97A-G128A-Y217Q-P239R A134T 200 G97A-G128A-Y217Q-N240K A134T 201 G97A-G128A-Y217Q-L267V A134T 202 G97A-G128A-Y217Q-A273S A134T 203 G97A-G128A-Y217Q-S159K A144K 204 G97A-G128A-Y217Q-S162K A144K 205 G97A-G128A-Y217Q-S161P A144K 206 G97A-G128A-Y217Q-V203Y A144K 207 G97A-G128A-Y217Q-G211T A144K 208 G97A-G128A-Y217Q-K213T A144K 209 G97A-G128A-Y217Q-P239R A144K 210 G97A-G128A-Y217Q-N240K A144K 211 G97A-G128A-Y217Q-L267V A144K 212 G97A-G128A-Y217Q-A273S A144K 213 G97A-G128A-Y217Q-S159K S145D 214 G97A-G128A-Y217Q-S162K S145D 215 G97A-G128A-Y217Q-S161P S145D 216 G97A-G128A-Y217Q-V203Y S145D 217 G97A-G128A-Y217Q-G211T S145D 218 G97A-G128A-Y217Q-K213T S145D 219 G97A-G128A-Y217Q-P239R S145D 220 G97A-G128A-Y217Q-N240K S145D 221 G97A-G128A-Y217Q-L267V S145D 222 G97A-G128A-Y217Q-A273S S145D 223 G97A-G128A-Y217Q-V203Y S159K 224 G97A-G128A-Y217Q-G211T S159K 225 G97A-G128A-Y217Q-K213T S159K 226 G97A-G128A-Y217Q-P239R S159K 227 G97A-G128A-Y217Q-N240K S159K 228 G97A-G128A-Y217Q-L267V S159K 229 G97A-G128A-Y217Q-A273S S159K 230 G97A-G128A-Y217Q-V203Y S162K 231 G97A-G128A-Y217Q-G211T S162K 232 G97A-G128A-Y217Q-K213T S162K 233 G97A-G128A-Y217Q-P239R S162K 234 G97A-G128A-Y217Q-N240K S162K 235 G97A-G128A-Y217Q-L267V S162K 236 G97A-G128A-Y217Q-A273S S162K 237 G97A-G128A-Y217Q-V203Y S161P 238 G97A-G128A-Y217Q-G211T S161P 239 G97A-G128A-Y217Q-K213T S161P 240 G97A-G128A-Y217Q-239R S161P 241 G97A-G128A-Y217Q-N240K S161P 242 G97A-G128A-Y217Q-L267V S161P 243 G97A-G128A-Y217Q-A273S S161P 244 G97A-G128A-Y217Q-G211T V203Y 245 G97A-G128A-Y217Q-K213T V203Y 246 G97A-G128A-Y217Q-P239R V203Y 247 G97A-G128A-Y217Q-N240K V203Y 248 G97A-G128A-Y217Q-L267V V203Y 249 G97A-G128A-Y217Q-A273S V203Y 250 G97A-G128A-Y217Q-P239R G211T 251 G97A-G128A-Y217Q-N240K G211T 252 G97A-G128A-Y217Q-L267V G211T 253 G97A-G128A-Y217Q-A273S G211T 254 G97A-G128A-Y217Q-P239R K213T 255 G97A-G128A-Y217Q-N240K K213T 256 G97A-G128A-Y217Q-L267V K213T 257 G97A-G128A-Y217Q-A273S K213T 258 G97A-G128A-Y217Q-L267V P239R 259 G97A-G128A-Y217Q-A273S P239R 260 G97A-G128A-Y217Q-L267V N240K 261 G97A-G128A-Y217Q-A273S N240K

Example 12 Table of Detergents

The compositions of the detergents used in the assays for Part I Example 12 are shown in Table 12-1. BPN′ variant protein samples were added to the detergent compositions as described in Part I Example 1 to assay for the various properties listed.

TABLE 12-1 Composition of Detergents Used in the Assays to Test BPN′ Variants Composition (wt % of Composition) Ingredient 1 2 3 C₁₂₋₁₅ Alkylethoxy(1.8)sulphate 14.7 11.6 C_(11.8) Alkylbenzene sulfonate 4.3 11.6 8.3 C₁₆₋₁₇ Branched alkyl sulphate 1.7 1.29 C₁₂₋₁₄ Alkyl-9-ethoxylate 0.9 1.07 C₁₂ dimethylamine oxide 0.6 0.64 Citric acid 3.5 0.65 3 C₁₂₋₁₈ fatty acid 1.5 2.32 3.6 Sodium Borate (Borax) 2.5 2.46 1.2 Sodium C₁₂₋₁₄ alkyl ethoxy 3 sulfate 2.9 C₁₄₋₁₅ alkyl 7-ethoxylate 4.2 C₁₂₋₁₄ Alkyl-7-ethoxylate 1.7 Ca formate 0.09 0.09 A compound having the following general structure: 1.2 bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n), wherein n = from 20 to 30, and x = from 3 to 8, or sulphated or sulphonated variants thereof Random graft co-polymer¹ 1.46 0.5 Ethoxylated Polyethylenimine ² 1.5 1.29 Diethylene triamine pentaacetic acid 0.34 0.64 Diethylene triamine penta(methylene phosphonic acid) 0.3 Tinopal AMS-GX 0.06 Tinopal CBS-X 0.2 0.17 Amphiphilic alkoxylated grease cleaning polymer ³ 1.28 1 0.4 Ethanol 2 1.58 1.6 Propylene Glycol 3.9 3.59 1.3 Diethylene glycol 1.05 1.54 Polyethylene glycol 0.06 0.04 Monoethanolamine 3.05 2.41 0.4 NaOH 2.44 1.8 Sodium Cumene Sulphonate 1 Sodium Formate 0.11 Water, Aesthetics (Dyes, perfumes) and Minors balance balance balance (Enzymes, solvents, structurants) ¹“Random graft copolymer” is a polyvinyl acetate grafted polyethylene oxide copolymer having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is about 6000 and the weight ratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60 and no more than 1 grafting point per 50 ethylene oxide units. ² Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH. ³ Amphiphilic alkoxylated grease cleaning polymer is a polyethylenimine (MW = 600) with 24 ethoxylate groups per —NH and 16 propoxylate groups per —NH.

Stain Removal Performance of BPN′ Combinatorial Variants

Experiments to evaluate the stain removal performance of BPN′ combinatorial variants generated as described in Example 11 were performed using BMI stained microswatches. The assay was performed as described in Example 1 (BMI microswatch assay). Table 12-2 provides Performance Index (PI) values of variants generated from RCL4 library using BMI microswatch assay in Detergent Composition 1 at pH 8 and 16° C. and Detergent Composition 1 at pH 8 and 32° C. and BMI microswatch assay in heat deactivated commercial TIDE® 2× Cold (Proctor & Gamble) detergent at 16° C. and pH 8. Heat inactivation of commercial detergent formulas serves to destroy the endogenous enzymatic activity of any protein components while retaining the properties of nonenzymatic components. Heat inactivation of the detergents was performed by placing pre-weighed amounts of liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The TIDE® 2× Cold detergent was purchased from local supermarket stores. Both unheated and heated detergents were assayed within 5 minutes of dissolving the detergent, in order to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay. Working solutions were made from the heat inactivated stock. Appropriate amounts of water hardness and buffer were added to the detergent solutions to match the desired conditions (Table 12-2). The solutions were mixed by vortexing or inverting the bottles.

TABLE 12-2 Working Detergent Solutions Temp Detergent Hardness Detergent (° C.) g/L pH Buffer Gpg TIDE ® 2X Cold 16, 32 0.98 8 5 mM HEPES 6

The sequences of the variants listed in Table 12-3 are relative to BPN′-v3: G97A-G128A-Y217Q. The PI values are calculated relative to BPN′-v3. All mutants in this list have a PI cutoff equal or greater than 0.5 for at least one property tested. “Det. Comp.” means Detergent Composition.

TABLE 12-3 Performance Index Values of Variants Generated from RCL4 Library Sequence Sequence Relative to TIDE ® Det. Comp. 1, Det. Comp. 1, Relative to BPN′-v3: G97A- Detergent pH 8, pH 8, 16° pH 8, 32° BPN′ G128A-Y217Q 16° C., BMI PI C., BMI PI C., BMI PI S87T-A88L-S89G- S87T-A88L- 1.00 1.12 0.99 G97A-G128A- S89G Y217Q N61P-S63H-G97A- N61P-S63H 1.03 1.12 1.01 G128A-Y217Q S87G-A88V-S89A- S87G-A88V- 1.02 1.11 0.99 G97A-G128A- S89A Y217Q P86S-S87G-A88V- P86S-S87G- 1.00 1.10 1.00 G97A-G128A- A88V Y217Q Q59S-N61P-G97A- Q59S-N61P 1.01 1.09 1.00 G128A-Y217Q S24G-N25G-G97A- S24G-N25G 0.99 1.09 1.02 G128A-Y217Q N61P-N62S-G97A- N61P-N62S 0.99 1.06 0.98 G128A-Y217Q G97A-G128A- P129Q-S130G- 0.96 1.06 0.99 P129Q-S130G- G131S G131S-Y217Q L75S-N76Y-G97A- L75S-N76Y 0.99 1.06 1.00 G128A-Y217Q G97A-G128A- V203Y 0.99 1.05 1.01 V203Y-Y217Q T55P-G97A-G128A- T55P 0.98 1.04 0.98 Y217Q A88V-L90I-G97A- A88V-L90I 0.99 1.04 1.00 G128A-Y217Q G97A-G128A- G211R-N212S- 0.97 1.04 0.98 G211R-N212S- K213V K213V-Y217Q G23A-S24G-N25G- G23A-S24G- 0.98 1.04 0.98 G97A-G128A- N25G Y217Q T22N-S24A-G97A- T22N-S24A 0.98 1.03 0.97 G128A-Y217Q S24R-G97A-G128A- S24R 0.95 1.02 0.99 Y217Q G97A-A98S-G128A- A98S 0.95 1.02 0.99 Y217Q BPN′-v3: G97A- BPN′-v3 1.00 1.00 1.00 G128A-Y217Q G97A-G128A- T158G-S159G 0.95 0.99 0.97 T158G-S159G- Y217Q Q59E-N61P-G97A- Q59E-N61P 0.90 0.94 0.90 G128A-Y217Q G97A-A98E-G128A- A98E 0.92 0.91 0.90 Y217Q

The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acid substitutions selected from those in Table 12-3, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Each such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Table 12-4 provides Performance Index (PI) values of variants generated from RCL 5-7 and FS1-3 using BMI microswatch assay in Detergent Composition 1 (from Table 1-3) at 16° C. and pH 8, BMI microswatch assay in Detergent Composition 2 (from Table 1-3) at 16° C. and pH 8, and stability measured in Detergent Composition 3 (from Table 1-3). PI values for specific activity by AAPF hydrolysis (Specific AAPF PI) were also determined. All assays were performed as described in Example 1. The sequences of the variants listed are relative to BPN′-v3: G97A-G128A-Y217Q. PI values were calculated relative to BPN′-v3. All mutants in this list have a PI cutoff equal or greater than 0.5 for at least one property tested. PI values less than 0.01 were modified to display 0.01 in bold italics.

TABLE 12-4 Performance Index Values of Variants Generated From RCL 5-7 and FS1-3 Sequence Source Sequence Relative to Det. Comp. 1, Det. Comp. 2, Det. Comp. 3, of Relative to BPN′-v3: G97A- pH 8, 16° C., pH 8, 16° C., Specific Stability Variants BPN′ G128A-Y217Q BMI PI BMI PI AAPF PI PI RCL6 G97A-G128A- P86S-S87G- 1.21 1.14 1.17 0.03 Y217Q-P86S- A88V-A116N- S87G-A88V- N117S-N118G A116N-N117S- N118G FS1 G97A-G128A- S24G-N25G- 1.05 1.12 1.96 1.00 Y217Q-S24G- N61P-S101N N25G-N61P- S101N FS1 G97A-G128A- S24G-N25G- 1.04 1.10 1.71 0.03 Y217Q-S24G- S53G-T55P- N25G-S53G- S87T-A88L- T55P-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y FS1 G97A-G128A- N61P-S78N- 1.03 1.10 1.73 0.33 Y217Q-N61P- S101N-V203Y S78N-S101N- V203Y FS1 G97A-G128A- T55P-N61P- 1.06 1.10 1.97 0.39 Y217Q-T55P- S78N-S101N- N61P-S78N- V203Y S101N-V203Y FS1 G97A-G128A- S53G-T55P- 1.03 1.10 2.22 0.33 Y217Q-S53G- N61P-S78N- T55P-N61P- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N RCL7 G97A-G128A- V203Y-L267V 1.10 1.09 1.12 0.09 Y217Q-V203Y- L267V FS1 G97A-G128A- S24G-N25G- 1.04 1.09 1.82 1.13 Y217Q-S24G- T55P-S101N N25G-T55P- S101N RCL7 G97A-G128A- A134T-L267V 1.13 1.08 0.93 0.66 Y217Q-A134T- L267V FS1 G97A-G128A- S24G-N25G- 1.01 1.08 1.10 0.29 Y217Q-S24G- S53G-T55P- N25G-S53G- N61P-S78N- T55P-N61P- S87T-A88L- S78N-S87T- S89G A88L-S89G FS1 G97A-G128A- S24G-N25G- 1.07 1.08 2.11 0.09 Y217Q-S24G- S53G-N61P- N25G-S53G- S101N-V203Y N61P-S101N- V203Y RCL6 G97A-G128A- N25Y-Q59S- 1.07 1.08 0.85 0.66 Y217Q-N25Y- N61P Q59S-N61P RCL7 G97A-G128A- I111V-S161P 1.10 1.08 0.66 0.98 Y217Q-I111V- S161P RCL7 G97A-G128A- I115V-L267V 1.10 1.08 1.07 0.63 Y217Q-I115V- L267V FS1 G97A-G128A- T55P-S78N- 0.99 1.08 1.39 0.16 Y217Q-T55P- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A- N25Y-P129Q- 1.07 1.08 0.88 0.67 Y217Q-N25Y- S130G-G131S- P129Q-S130G- A137T G131S-A137T RCL7 G97A-G128A- N61P-S63H- 1.04 1.08 1.59 0.78 Y217Q-N61P- A128S-P129Q S63H-A128S- P129Q FS1 G97A-G128A- S53G-N61P- 1.03 1.08 1.91 0.09 Y217Q-S53G- S101N-V203Y N61P-S101N- V203Y FS1 G97A-G128A- S24G-N25G- 1.08 1.07 1.44 0.33 Y217Q-S24G- S53G-S78N- N25G-S53G- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N FS1 G97A-G128A- N61P-S78N- 1.07 1.07 1.80 0.31 Y217Q-N61P- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N RCL6 G97A-G128A- N25Y-N61P- 1.06 1.07 0.74 0.60 Y217Q-N25Y- S63H N61P-S63H RCL5 G97A-G128A- Q59S-N61P- 0.99 1.07 0.69 0.09 Y217Q-Q59S- V203Y N61P-V203Y RCL6 G97A-G128A- V8L-N25Y- 1.08 1.07 1.22 0.03 Y217Q-V8L- P129Q-S130G- N25Y-P129Q- G131S S130G-G131S RCL6 G97A-G128A- P86S-S87G- 1.16 1.07 1.26 0.03 Y217Q-P86S- A88V-P239R S87G-A88V- P239R FS1 G97A-G128A- S24G-N25G- 1.02 1.07 2.07 0.11 Y217Q-S24G- S53G-T55P- N25G-S53G- N61P-S101N- T55P-N61P- V203Y S101N-V203Y RCL5 G97A-G128A- S24G-N25G- 1.09 1.06 1.25 1.00 Y217Q-S24G- P129Q-S130G- N25G-P129Q- G131S S130G-G131S FS3 G97A-G128A- N240K 1.04 1.06 0.90 0.94 Y217Q-N240K RCL5 G97A-G128A- G23A-S24G- 1.06 1.06 1.05 0.06 Y217Q-G23A- N25G-G211R- S24G-N25G- N212S-K213V G211R-N212S- K213V RCL7 G97A-G128A- N61P-S63H- 1.03 1.06 0.43 1.14 Y217Q-N61P- S78N-I111V- S63H-S78N- A134T I111V-A134T RCL6 G97A-G128A- S63T-P86S- 1.17 1.06 1.46 0.02 Y217Q-S63T- S87G-A88V P86S-S87G- A88V RCL6 G97A-G128A- G23A-S24G- 1.08 1.06 0.91 0.21 Y217Q-G23A- N25G-A116N- S24G-N25G- N117S-N118G A116N-N117S- N118G FS1 G97A-G128A- S78N-S87T- 1.03 1.06 1.40 0.33 Y217Q-S78N- A88L-S89G- S87T-A88L- S101N S89G-S101N RCL6 G97A-G128A- S24G-N25G- 1.06 1.06 0.74 1.05 Y217Q-S24G- A116N-N117S- N25G-A116N- N118G N117S-N118G RCL7 G97A-G128A- T55P-N240K 1.02 1.06 0.91 0.93 Y217Q-T55P- N240K RCL6 G97A-G128A- T55P-P129V- 1.07 1.06 0.85 0.86 Y217Q-T55P- P194S P129V-P194S RCL6 G97A-G128A- N25Y-S87G- 1.07 1.05 1.14 0.08 Y217Q-N25Y- A88V-S89A S87G-A88V- S89A FS1 G97A-G128A- S24G-N25G- 1.02 1.05 1.19 0.06 Y217Q-S24G- S87T-A88L- N25G-S87T- S89G-S101N A88L-S89G- S101N RCL7 G97A-G128A- P129Q-S130G- 1.04 1.05 0.94 0.05 Y217Q-P129Q- G131S-V203Y S130G-G131S- V203Y RCL6 G97A-G128A- Q59S-N61P- 1.05 1.05 0.80 0.84 Y217Q-Q59S- N240K N61P-N240K FS3 G97A-G128A- S24R-P40E- 1.13 1.05 1.08 1.31 Y217Q-S24R- P129E-S159K- P40E-P129E- K265R S159K-K265R RCL7 G97A-G128A- P52S-T55P- 1.09 1.05 0.53 0.15 Y217Q-P52S- V203Y T55P-V203Y RCL6 G97A-G128A- S24R-P129E 1.11 1.05 0.88 0.59 Y217Q-S24R- P129E FS1 G97A-G128A- S24G-N25G- 0.98 1.05 1.17 1.16 Y217Q-S24G- S53G-N61P- N25G-S53G- S78N N61P-S78N FS1 G97A-G128A- S24G-N25G- 1.07 1.05 1.71 1.29 Y217Q-S24G- T55P-S78N- N25G-T55P- S101N S78N-S101N RCL6 G97A-G128A- P86S-S87G- 1.10 1.05 0.76 0.05 Y217Q-P86S- A88V-A116S- S87G-A88V- N117G-N118R A116S-N117G- N118R RCL6 G97A-G128A- N61P-S87T- 1.00 1.05 1.12 0.05 Y217Q-N61P- A88L-S89G S87T-A88L- S89G FS1 G97A-G128A- S24G-N25G- 0.96 1.05 0.96 0.29 Y217Q-S24G- S53G-T55P- N25G-S53G- S78N-S87T- T55P-S78N- A88L-S89G S87T-A88L- S89G RCL5 G97A-G128A- G23A-S24G- 1.06 1.05 0.89 0.13 Y217Q-G23A- N25G-N61P- S24G-N25G- S63H N61P-S63H RCL6 G97A-G128A- S24R-Q59S- 1.07 1.05 0.94 0.49 Y217Q-S24R- N61P Q59S-N61P RCL6 G97A-G128A- N61P-P129Q- 1.07 1.05 1.13 0.78 Y217Q-N61P- S130G-G131S P129Q-S130G- G131S FS1 G97A-G128A- S24G-N25G- 1.05 1.04 1.98 0.32 Y217Q-S24G- S53G-T55P- N25G-S53G- N61P-S78N- T55P-N61P- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N FS1 G97A-G128A- S24G-N25G- 1.05 1.04 1.57 0.15 Y217Q-S24G- S53G-T55P- N25G-S53G- S101N-V203Y T55P-S101N- V203Y FS1 G97A-G128A- N61P-S78N- 1.02 1.04 1.60 0.12 Y217Q-N61P- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y FS1 G97A-G128A- S24G-N25G- 1.03 1.04 2.01 1.27 Y217Q-S24G- S53G-T55P- N25G-S53G- S78N-S101N T55P-S78N- S101N FS1 G97A-G128A- S24G-N25G- 1.01 1.04 1.77 0.07 Y217Q-S24G- S53G-S101N- N25G-S53G- V203Y S101N-V203Y FS1 G97A-G128A- S24G-N25G- 1.09 1.04 1.54 0.28 Y217Q-S24G- S78N-S101N- N25G-S78N- V203Y S101N-V203Y RCL7 G97A-G128A- P129Q-S130G- 1.14 1.04 1.37 0.56 Y217Q-P129Q- G131S-A133V- S130G-G131S- L267V A133V-L267V FS1 G97A-G128A- S87T-A88L- 1.05 1.04 1.44 0.07 Y217Q-S87T- S89G-S101N A88L-S89G- S101N RCL6 G97A-G128A- G23A-S24G- 1.07 1.04 0.92 0.16 Y217Q-G23A- N25G-P239R S24G-N25G- P239R RCL6 G97A-G128A- S87G-A88V- 1.07 1.04 0.92 0.37 Y217Q-S87G- S89A-A116N- A88V-S89A- N117S-N118G A116N-N117S- N118G RCL6 G97A-G128A- Q59S-N61P- 1.09 1.04 0.68 0.91 Y217Q-Q59S- A116S-N117G- N61P-A116S- N118R N117G-N118R RCL5 G97A-G128A- Q59S-N61P- 1.03 1.04 0.81 0.08 Y217Q-Q59S- S87T-A88L- N61P-S87T- S89G A88L-S89G FS1 G97A-G128A- S24G-N25G- 0.96 1.04 0.73 0.04 Y217Q-S24G- S53G-S87T- N25G-S53G- A88L-S89G- S87T-A88L- V203Y S89G-V203Y RCL7 G97A-G128A- A134T-G211T 1.11 1.04 0.71 0.35 Y217Q-A134T- G211T RCL7 G97A-G128A- T55P-A128S- 1.03 1.04 2.71 1.02 Y217Q-T55P- P129Q A128S-P129Q FS1 G97A-G128A- T55P-S78N- 1.05 1.04 1.56 0.33 Y217Q-T55P- S87T-A88L- S78N-S87T- S89G-S101N A88L-S89G- S101N RCL6 G97A-G128A- P86S-S87G- 1.09 1.04 0.83 0.04 Y217Q-P86S- A88V-T242R S87G-A88V- T242R RCL7 G97A-G128A- S161P-V203Y 1.04 1.04 0.81 0.09 Y217Q-S161P- V203Y FS1 G97A-G128A- S24G-N25G- 1.05 1.04 2.01 0.44 Y217Q-S24G- T55P-N61P- N25G-T55P- S78N-S101N- N61P-S78N- V203Y S101N-V203Y RCL7 G97A-G128A- G211T-L267V 1.08 1.03 1.16 0.30 Y217Q-G211T- L267V RCL6 G97A-G128A- P40E-T55P- 1.08 1.03 0.71 0.05 Y217Q-P40E- N269K T55P-N269K RCL6 G97A-G128A- S24R-A128S- 1.08 1.03 1.63 0.64 Y217Q-S24R- P129G A128S-P129G RCL6 G97A-G128A- S24G-N25G- 1.15 1.03 3.06 0.35 Y217Q-S24G- N61P-N62S- N25G-N61P- P194L-A232T N62S-P194L- A232T RCL6 G97A-G128A- T55P-A116S- 1.07 1.03 0.83 1.06 Y217Q-T55P- N117G-N118R A116S-N117G- N118R FS1 G97A-G128A- S24G-N25G- 1.03 1.03 1.76 0.32 Y217Q-S24G- S53G-S78N- N25G-S53G- S101N-V203Y S78N-S101N- V203Y RCL7 G97A-G128A- P129Q-S130G- 1.08 1.03 0.90 0.83 Y217Q-P129Q- G131S-N240K S130G-G131S- N240K FS1 G97A-G128A- S53G-T55P- 0.93 1.03 1.00 0.33 Y217Q-S53G- N61P-S78N- T55P-N61P- S87T-A88L- S78N-S87T- S89G A88L-S89G RCL6 G97A-G128A- N25Y-P129Q- 1.08 1.03 0.93 0.69 Y217Q-N25Y- S130G-G131S P129Q-S130G- G131S RCL6 G97A-G128A- T55P-I115V 1.06 1.03 1.07 1.07 Y217Q-T55P- I115V RCL6 G97A-G128A- N25Y-T55P 1.05 1.03 1.00 0.80 Y217Q-N25Y- T55P RCL6 G97A-G128A- G23A-S24G- 0.97 1.03 1.75 0.15 Y217Q-G23A- N25G-A128S- S24G-N25G- P129D A128S-P129D FS1 G97A-G128A- S53G-S78N- 1.00 1.03 1.68 0.13 Y217Q-S53G- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- P129S-V203Y S101N-P129S- V203Y RCL7 G97A-G128A- T55P-A134T 1.06 1.03 0.94 1.05 Y217Q-T55P- A134T RCL7 G97A-G128A- N61P-S63H- 1.03 1.02 0.49 1.18 Y217Q-N61P- S78N-I111V S63H-S78N- I111V FS2 Y217Q-N61P- N61P-A97G- 0.98 1.02 NA NA A97G-G102A- G102A-A128G- A128G-P129S P129S FS1 G97A-G128A- S53G-N61P- 1.02 1.02 2.08 0.88 Y217Q-S53G- S101N N61P-S101N RCL5 G97A-G128A- Q59S-N61P- 1.01 1.02 0.90 0.20 Y217Q-Q59S- S87G-A88V- N61P-S87G- S89A A88V-S89A FS1 G97A-G128A- S53G-S87T- 0.96 1.02 1.11 0.04 Y217Q-S53G- A88L-S89G- S87T-A88L- S101N-V203Y S89G-S101N- V203Y RCL6 G97A-G128A- S87T-A88L- 1.06 1.02 1.21 0.10 Y217Q-S87T- S89G-P129S A88L-S89G- P129S FS1 G97A-G128A- S53G-T55P- 1.03 1.02 1.70 0.48 Y217Q-S53G- S78N-S101N- T55P-S78N- V203Y S101N-V203Y RCL5 G97A-G128A- T55P-P129Q- 1.01 1.02 1.18 0.91 Y217Q-T55P- S130G-G131S P129Q-S130G- G131S RCL5 G97A-G128A- Q59S-N61P- 0.98 1.02 0.83 0.80 Y217Q-Q59S- P129Q-S130G- N61P-P129Q- G131S S130G-G131S RCL7 G97A-G128A- A134T-P239R 1.03 1.02 0.53 0.98 Y217Q-A134T- P239R RCL5 G97A-G128A- T55P-V203Y 1.01 1.01 1.02 0.23 Y217Q-T55P- V203Y RCL7 G97A-G128A- T55P-S78N- 1.03 1.01 1.05 1.25 Y217Q-T55P- S89Y S78N-S89Y RCL5 G97A-G128A- T22N-S24A- 1.00 1.01 0.69 0.46 Y217Q-T22N- N61P-S63H S24A-N61P- S63H RCL7 G97A-G128A- S161P-L267V 1.05 1.01 1.00 0.66 Y217Q-S161P- L267V RCL6 G97A-G128A- T55P-L75H- 1.06 1.01 0.69 0.59 Y217Q-T55P- N76G L75H-N76G RCL7 G97A-G128A- A134T-S161P 1.07 1.01 0.73 1.00 Y217Q-A134T- S161P RCL7 G97A-G128A- S87T-A88L- 1.08 1.01 0.66 0.13 Y217Q-S87T- S89G-A134T A88L-S89G- A134T RCL6 G97A-G128A- T55P-A116N- 1.06 1.01 1.06 1.11 Y217Q-T55P- N117S-N118G A116N-N117S- N118G v12 G97A-G128S- A128S 1.02 1.01 1.65 1.00 Y217Q RCL7 G97A-G128A- T55P-S78N- 1.07 1.00 1.00 1.32 Y217Q-T55P- I115V S78N-I115V RCL6 G97A-G128A- Y6Q-P129Q- 1.03 1.00 0.98 0.23 Y217Q-Y6Q- S130G-G131S P129Q-S130G- G131S RCL7 G97A-G128A- S24R-P129Q- 1.06 1.00 1.11 0.61 Y217Q-S24R- S130G-G131S P129Q-S130G- G131S FS1 G97A-G128A- S24G-N25G- 0.99 1.00 1.61 1.21 Y217Q-S24G- S53G-S78N- N25G-S53G- S101N S78N-S101N RCL6 G97A-G128A- T55P-P129V 1.07 1.00 0.70 1.00 Y217Q-T55P- P129V v3 G97A-G128A- BPN′-v3 1.00 1.00 1.00 1.00 Y217Q FS2 G97A-Y217Q- N61P-N62Q- 0.95 1.00 NA NA N61P-N62Q- G100N-A128G G100N-A128G RCL6 G97A-G128A- T55P-P129Q 1.10 1.00 1.77 1.15 Y217Q-T55P- P129Q FS1 G97A-G128A- S24G-N25G- 1.05 1.00 1.84 0.12 Y217Q-S24G- S53G-T55P- N25G-S53G- N61P-S78N- T55P-N61P- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A- S87T-A88L- 1.04 1.00 0.83 0.10 Y217Q-S87T- S89G-N240K A88L-S89G- N240K RCL7 G97A-G128A- A134T-N240K 1.07 0.99 0.61 0.96 Y217Q-A134T- N240K RCL7 G97A-G128A- S87T-A88L- 1.04 0.99 0.68 0.14 Y217Q-S87T- S89G-P239R A88L-S89G- P239R RCL7 G97A-G128A- P129Q-S130G- 1.08 0.99 1.35 0.61 Y217Q-P129Q- G131S-L267V S130G-G131S- L267V RCL7 G97A-G128A- P129Q-N240K 1.06 0.99 1.39 1.00 Y217Q-P129Q- N240K FS1 G97A-G128A- S78N-S87T- 0.93 0.99 0.74 0.15 Y217Q-S78N- A88L-S89G- S87T-A88L- V203Y S89G-V203Y RCL7 G97A-G128A- I111V-A273S 1.00 0.99 0.48 0.27 Y217Q-I111V- A273S FS1 G97A-G128A- S24G-N25G- 1.03 0.99 2.17 0.71 Y217Q-S24G- T55P-S78N- N25G-T55P- A88V-S101N S78N-A88V- S101N FS1 G97A-G128A- S24G-N25G- 0.96 0.98 1.15 1.20 Y217Q-S24G- T55P-S78N N25G-T55P- S78N FS1 G97A-G128A- S24G-N25G- 1.02 0.98 1.27 0.10 Y217Q-S24G- S53G-S78N- N25G-S53G- S87T-A88L- S78N-S87T- S101N-V203Y A88L-S101N- V203Y FS1 G97A-G128A- S24G-N25G- 0.97 0.98 0.75 0.14 Y217Q-S24G- S53G-S78N- N25G-S53G- S87T-A88L- S78N-S87T- S89G-V203Y A88L-S89G- V203Y FS1 G97A-G128A- S24G-N25G- 1.06 0.98 1.21 0.14 Y217Q-S24G- S53G-S78N- N25G-S53G- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL5 G97A-G128A- S87G-A88V- 1.01 0.98 1.18 0.29 Y217Q-S87G- S89A-P129Q- A88V-S89A- S130G-G131S P129Q-S130G- G131S RCL7 G97A-G128A- N61P-S63H- 1.03 0.98 0.73 1.16 Y217Q-N61P- S78N-S161P S63H-S78N- S161P FS1 G97A-G128A- T55P-N61P- 1.04 0.98 1.75 0.14 Y217Q-T55P- S78N-S87T- N61P-S78N- A88L-S89G- S87T-A88L- S101N-V203Y S89G-S101N- V203Y RCL7 G97A-G128A- I111V-P129Q- 1.00 0.98 0.52 0.97 Y217Q-I111V- S130G-G131S P129Q-S130G- G131S RCL5 G97A-G128A- T22N-S24A- 0.96 0.98 0.94 0.64 Y217Q-T22N- T55P S24A-T55P RCL7 G97A-G128A- I115V-N240K 1.04 0.98 0.66 0.90 Y217Q-I115V- N240K RCL6 G97A-G128A- S87G-A88V- 0.98 0.98 0.66 0.40 Y217Q-S87G- S89A-A116N- A88V-S89A- N117S-N118G- A116N-N117S- P172H N118G-P172H FS1 G97A-G128A- S24G-N25G- 1.01 0.98 1.28 0.31 Y217Q-S24G- S78N-S87T- N25G-S78N- A88L-S89G- S87T-A88L- S101N S89G-S101N RCL6 G97A-G128A- S24G-N25G- 1.03 0.98 0.59 1.13 Y217Q-S24G- I115V-A134T N25G-I115V- A134T RCL6 G97A-G128A- T55P-A128S- 0.97 0.97 1.39 1.00 Y217Q-T55P- P129D A128S-P129D RCL7 G97A-G128A- I111V-S159K 0.99 0.97 0.64 1.12 Y217Q-I111V- S159K RCL7 G97A-G128A- N240K-A273S 1.03 0.96 0.64 0.19 Y217Q-N240K- A273S RCL7 G97A-G128A- S159K-L267V 1.00 0.96 1.08 0.79 Y217Q-S159K- L267V RCL7 G97A-G128A- I111V-P129Q- 1.01 0.96 0.83 0.42 Y217Q-I111V- G211T P129Q-G211T RCL7 G97A-G128A- I115V-A273S 1.05 0.95 0.74 0.20 Y217Q-I115V- A273S RCL6 G97A-G128A- S89Y 0.99 0.95 0.70 0.88 Y217Q-S89Y RCL6 G97A-G128A- S24R-A116N- 1.08 0.95 0.91 0.60 Y217Q-S24R- N117S-N118G A116N-N117S- N118G RCL7 G97A-G128A- N61E-A144K 1.03 0.95 0.97 1.10 Y217Q-N61E- A144K RCL6 G97A-G128A- P129Q-S130G- 1.02 0.95 0.85 0.99 Y217Q-P129Q- G131S-P239R S130G-G131S- P239R RCL7 G97A-G128A- S87T-A88L- 0.97 0.95 0.59 0.13 Y217Q-S87T- S89G-I115V A88L-S89G- I115V RCL6 G97A-G128A- T55P-A92G 0.96 0.94 0.56 0.91 Y217Q-T55P- A92G FS3 G97A-G128A- S145D-S159K- 0.98 0.94 0.76 1.08 Y217Q-S145D- N240K-Q275E S159K-N240K- Q275E RCL7 G97A-G128A- S89Y-P129Q- 1.04 0.94 0.71 0.70 Y217Q-S89Y- S130G-G131S P129Q-S130G- G131S RCL7 G97A-G128A- P129Q-S130G- 1.01 0.94 1.00 0.93 Y217Q-P129Q- G131S-S162K S130G-G131S- S162K RCL7 G97A-G128A- I111V-A134T 0.98 0.94 0.37 1.06 Y217Q-I111V- A134T RCL6 G97A-G128A- P40E-S53Y- 0.99 0.94 0.97 0.35 Y217Q-P40E- S78Y-P86S- S53Y-S78Y- S87G-A88V P86S-S87G- A88V RCL6 G97A-G128A- S24G-N25G- 0.93 0.93 0.58 0.52 Y217Q-S24G- L75H-N76G N25G-L75H- N76G FS2 G97A-Y217Q- N61P-A128G- 0.85 0.93 0.65 0.92 N61P-A128G- P129S-S130P P129S-S130P RCL6 G97A-G128A- S24R-S145D 0.99 0.93 0.89 0.59 Y217Q-S24R- S145D FS3 G97A-G128A- S24R-S145D- 0.92 0.92 0.63 0.68 Y217Q-S24R- P239R-Q275E S145D-P239R- Q275E RCL7 G97A-G128A- S24R-S78N- 0.95 0.92 1.16 0.55 Y217Q-S24R- S182P-L267V S78N-S182P- L267V FS1 G97A-G128A- S53G-N61P- 1.04 0.92 1.67 0.02 Y217Q-S53G- S87T-A88L- N61P-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A- P5S-S87G- 0.98 0.92 0.64 0.07 Y217Q-P5S- A88V-S89A- S87G-A88V- A116G-N117R S89A-A116G- N117R FS1 G97A-G128A- S53G-N61P- 0.99 0.92 1.65 0.11 Y217Q-S53G- S78N-S87T- N61P-S78N- A88L-S89G- S87T-A88L- S101N-V203Y S89G-S101N- V203Y RCL6 G97A-G128A- Q59S-N61P- 0.93 0.92 0.42 1.04 Y217Q-Q59S- A116N-N117S- N61P-A116N- N118G N117S-N118G RCL7 G97A-G128A- P239R-A273S 0.94 0.91 0.52 0.23 Y217Q-P239R- A273S FS1 G97A-G128A- S53G-S78N- 0.94 0.91 1.16 0.13 Y217Q-S53G- S87T-A88L- S78N-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A- S24R-P129V 0.98 0.91 0.52 0.58 Y217Q-S24R- P129V RCL7 G97A-G128A- I111V-P239R 0.97 0.91 0.38 1.08 Y217Q-I111V- P239R FS1 G97A-G128A- S87T-A88L- 0.90 0.91 0.79 0.05 Y217Q-S87T- S89G-S101N- A88L-S89G- V203Y S101N-V203Y RCL6 G97A-G128A- T55P-P129L 0.99 0.91 0.62 0.98 Y217Q-T55P- P129L RCL7 G97A-G128A- S87T-A88L- 0.92 0.90 0.42 0.13 Y217Q-S87T- S89G-I111V A88L-S89G- I111V RCL7 G97A-G128A- S145D-A273S 0.91 0.90 0.66 0.17 Y217Q-S145D- A273S RCL6 G97A-G128A- P129Q-S130G- 0.94 0.90 0.51 0.89 Y217Q-P129Q- G131S-T242R S130G-G131S- T242R RCL7 G97A-G128A- S3F-S87T- 0.93 0.89 0.55 0.07 Y217Q-S3F- A88L-S89G- S87T-A88L- G211T S89G-G211T RCL6 G97A-G128A- S87G-A88V- 0.97 0.89 1.21 0.37 Y217Q-S87G- S89A-S162K A88V-S89A- S162K RCL7 G97A-G128A- S89Y-G211T 0.89 0.88 0.53 0.41 Y217Q-S89Y- G211T RCL7 G97A-G128A- S87T-A88L- 0.93 0.88 0.74 0.12 Y217Q-S87T- S89G-A144K A88L-S89G- A144K RCL6 G97A-G128A- P129Q-S130G- 0.95 0.88 1.21 0.96 Y217Q-P129Q- G131S-S159K S130G-G131S- S159K RCL6 G97A-G128A- A116N-N117S- 0.90 0.88 0.54 0.95 Y217Q-A116N- N118G-P129Q- N117S-N118G- S130G-G131S P129Q-S130G- G131S RCL6 G97A-G128A- S24G-N25G- 0.86 0.87 0.44 1.02 Y217Q-S24G- P129V N25G-P129V FS1 G97A-G128A- S24G-N25G- 0.87 0.86 0.83 0.12 Y217Q-S24G- S78N-S87T- N25G-S78N- A88L-S89G- S87T-A88L- S101N-V203Y S89G-S101N- V203Y FS2 G97A-Y217Q- N123G-A128G 0.83 0.86 1.00 0.14 N123G-A128G FS2 G97A-G128A- N61P-N62Q- 0.82 0.86 5.52 0.40 Y217Q-N61P- G100N-G102A- N62Q-G100N- M124I G102A-M124I RCL6 G97A-G128A- S24G-N25G- 0.90 0.85 0.65 1.10 Y217Q-S24G- K141E-T242R N25G-K141E- T242R RCL6 G97A-G128A- S87G-A88V- 0.89 0.85 0.44 0.32 Y217Q-S87G- S89A-A116N- A88V-S89A- N117S-N118G- A116N-N117S- A144T N118G-A144T FS1 G97A-G128A- T55P-N61P- 0.83 0.85 0.92 0.30 Y217Q-T55P- S87T-A88L- N61P-S87T- S89G-G110C- A88L-S89G- S130P G110C-S130P RCL6 G97A-G128A- L75S-N76Y- 0.84 0.83 0.41 0.13 Y217Q-L75S- A116S-N117G- N76Y-A116S- N118R N117G-N118R FS3 G97A-G128A- S145D-S159K- 0.74 0.81 0.38 0.65 Y217Q-S145D- K213L-P239R- S159K-K213L- N240K P239R-N240K RCL6 G97A-G128A- S24R-S87T- 0.86 0.80 0.53 0.08 Y217Q-S24R- A88L-S89G S87T-A88L- S89G RCL6 G97A-G128A- G23A-S24G- 0.88 0.79 0.53 0.20 Y217Q-G23A- N25G-P129V S24G-N25G- P129V RCL7 G97A-G128A- A134T-K213L 0.87 0.79 0.51 0.81 Y217Q-A134T- K213L RCL7 G97A-G128A- S89Y-A273S 0.81 0.79 0.48 0.19 Y217Q-S89Y- A273S RCL7 G97A-G128A- S24R-P239R 0.90 0.78 0.71 0.69 Y217Q-S24R- P239R FS2 G97A-Y217Q- N123G-A128G- 0.76 0.78 0.71 0.12 N123G-A128G- P129S P129S RCL7 G97A-G128A- S89Y-P239R 0.77 0.76 0.38 0.90 Y217Q-S89Y- P239R RCL6 G97A-G128A- S24G-N25G- 0.71 0.76 0.25 0.59 Y217Q-S24G- A92G N25G-A92G RCL6 G97A-G128A- N61P-S63H- 0.73 0.74 0.24 0.42 Y217Q-N61P- I115V-A228V S63H-I115V- A228V FNA Y217L A97G-A128G- 0.73 0.74 1.81 1.21 Q217L RCL6 G97A-G128A- L75S-N76Y- 0.73 0.73 0.34 0.09 Y217Q-L75S- P129V N76Y-P129V RCL6 G97A-G128A- S24R-P129L 0.80 0.73 0.35 0.58 Y217Q-S24R- P129L RCL6 G97A-G128A- S87G-A88V- 0.69 0.73 0.90 0.36 Y217Q-S87G- S89A-P129Q- A88V-S89A- S182Y-S204Y- P129Q-S182Y- P239Q S204Y-P239Q RCL6 G97A-G128A- S24R-A92G 0.75 0.73 0.28 0.44 Y217Q-S24R- A92G RCL6 G97A-G128A- S24R-A116S- 0.81 0.72 0.48 0.58 Y217Q-S24R- N117G-N118R A116S-N117G- N118R RCL6 G97A-G128A- G23A-S24G- 0.85 0.66 0.63 0.20 Y217Q-G23A- N25G-A116G- S24G-N25G- N117R A116G-N117R RCL6 G97A-G128A- S24G-N25G- 0.72 0.66 0.39 1.03 Y217Q-S24G- P129L N25G-P129L RCL6 G97A-G128A- S87T-A88L- 0.78 0.66 0.65 0.11 Y217Q-S87T- S89G-S101G A88L-S89G- S101G RCL6 G97A-G128A- G23A-S24G- 0.52 0.59 0.37 0.20 Y217Q-G23A- N25G-P129L S24G-N25G- P129L FS1 G97A-G128A- S53G-N61P- 0.55 0.58 0.29 0.16 Y217Q-S53G- G102A-V203Y N61P-G102A- V203Y RCL6 G97A-G128A- T55P-V147P 0.56 0.57 0.22 1.09 Y217Q-T55P- V147P RCL6 G97A-G128A- Y6Q-L75S- 0.55 0.52 0.24 0.17 Y217Q-Y6Q- N76Y L75S-N76Y RCL6 G97A-G128A- N61P-S63H- 0.36 0.37 0.14 0.95 Y217Q-N61P- V147P S63H-V147P RCL6 G97A-G128A- S24R-V147P 0.16 0.22 0.08 0.85 Y217Q-S24R- V147P RCL6 G97A-G128A- S24G-N25G- 0.21 0.18 0.06 0.71 Y217Q-S24G- V68C-A69G N25G-V68C- A69G FS1 G97A-G128A- S24G-N25G- 0.19 0.18 0.08 0.38 Y217Q-S24G- N61P-L75S- N25G-N61P- N76Y-S101N- L75S-N76Y- V203Y S101N-V203Y FS1 G97A-G128A- L75S-N76Y- 0.13 0.12 0.05 0.72 Y217Q-L75S- S78N-S101N- N76Y-S78N- V203Y S101N-V203Y FS1 G97A-G128A- L75S-N76Y- 0.04 0.04 0.07 0.73 Y217Q-L75S- S78N-S87T- N76Y-S78N- A88L-S89G- S87T-A88L- S101N-S130P S89G-S101N- S130P FS1 G97A-G128A- S24G-N25G- 0.06 0.04 0.06 0.33 Y217Q-S24G- S53G-S101N- N25G-S53G- S130P-V203Y S101N-S130P- V203Y RCL6 G97A-G128A- G47E-M50I-

0.03 0.02 1.21 Y217Q-G47E- L75S-N76Y- M50I-L75S- S162K N76Y-S162K FS1 G97A-G128A- S53G-T55P-

0.02 0.05 0.45 Y217Q-S53G- S87T-A88L- T55P-S87T- S89G-S101N- A88L-S89G- S130P-V203Y S101N-S130P- V203Y FS1 G97A-G128A- S24G-N25G-

0.02 1.21 Y217Q-S24G- L75S-N76Y- N25G-L75S- A128T-P129T- N76Y-A128T- S130G-G131Q- P129T-S130G- S132C-A133G- G131Q-S132C- A134T A133G-A134T RCL6 G97A-G128A- P129Q-S130G-

0.03 1.11 Y217Q-P129Q- G131S-V147P S130G-G131S- V147P FS1 G97A-G128A- S53G-T55P-

0.02 1.05 Y217Q-S53G- N61P-L75S- T55P-N61P- N76Y-S87T- L75S-N76Y- A88L-S89G- S87T-A88L- G102A-S130P- S89G-G102A- V203Y S130P-V203Y FS1 G97A-G128A- S53G-T55P- 0.03

0.02 1.03 Y217Q-S53G- N61P-L75S- T55P-N61P- N76Y-S101N- L75S-N76Y- S130P-V203Y S101N-S130P- V203Y

The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acid substitutions selected from those listed in Table 12-4, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Each such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Table 12-5 provides the Performance Index (PI) values of BPN′ variants (generated as described in “Generation of Variants to Improve BPN′ Stability”; see Table 11-3) for stain removal in BMI microswatch assay in Detergent Composition 1 at 16° C. and pH 8 (Det. Comp. 1, pH 8, 16° C., BMI PI) and for stability in LAS/EDTA (LAS/EDTA Stability PI). Assays were performed as described in Example 1 (BMI microswatch assay, LAS/EDTA stability assay). The sequences of the variants are shown relative to both BPN′ and FNA. That is, each variant sequence is the BPN′ or FNA sequence with the specified variant amino acid substitutions. PI values are shown relative to FNA parent, which is BPN′-Y217L.

TABLE 12-5 Performance Index of Stability-Improved BPN′ Variants Sequence Relative to Det. Comp. LAS/EDTA FNA: BPN′ Sequence 1 pH 8, 16° Stability Y217L Relative to BPN′ C., BMI PI PI P40E-S78N- P40E-S78N-S87D- 0.71 11.65 S87D Y217L P40E P40E-Y217L 0.96 8.33 T22V-S78N- T22V-S78N-Q206E- 0.75 5.95 Q206E-K213N K213N-Y217L T22V-S78N- T22V-S78N-K213N- 0.86 5.71 K213N Y217L S87D S87D-Y217L 0.90 4.04 S78N S78N-Y217L 0.87 3.86 K213N K213N-Y217L 0.91 1.84 Q206E Q206E-Y217L 0.86 1.76 T22V T22V-Y217L 0.97 1.46 FNA Y217L 1.00 1.00

The invention includes a protease variant having proteolytic activity and/or improved stability relative to FNA, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acid substitutions selected from those listed in Table 12-5, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Each such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Table 12-6 provides the Performance Index (PI) values of BPN′ variants generated from Library Parent: BPN′-v3: G97A-G128A-Y217Q (as described in “Generation of Variants to Improve BPN′ Stability”; see Table 11-3) for stain removal in a BMI microswatch assay in Detergent Composition 1 at 16° C. and pH 8 and for stability in LAS/EDTA. Assays were performed as described in Example 1 (BMI microswatch assay and LAS/EDTA stability assay). The Performance Index was calculated relative to BPN′-v3: G97A-G128A-Y217Q. All mutants in this list have a PI cutoff equal or greater than 0.5 for at least one property tested.

TABLE 12-6 Performance Index of Stability-Improved BPN′ Variants Sequence Det. Comp. LAS/ Relative to 1 pH 8, EDTA BPN′-v3: BPN′ Sequence 16° C., Stability G97A-G128A-Y217Q Relative to BPN′ BMI PI PI S87D-N76D-S78N S87D-N76D-S78N- 0.62 2.27 G97A-G128A-Y217Q P40E-S78N-S87D P40E-S78N-S87D- 0.21 2.18 G97A-G128A-Y217Q P40E-S87D P40E-S87D-G97A- 0.18 2.14 G128A-Y217Q S78N-P40E S78N-P40E-G97A- 0.80 2.03 G128A-Y217Q S87D-N76D S87D-N76D-G97A- 0.55 1.89 G128A-Y217Q P40E P40E-G97A-G128A- 0.84 1.79 Y217Q S78N-S87D S78N-S87D-G97A- 0.79 1.71 G128A-Y217Q S87D S87D-G97A-G128A- 0.78 1.20 Y217Q S78N S78N-G97A-G128A- 0.93 1.14 Y217Q BPN′-v3 G97A-G128A-Y217Q 1.00 1.00

The invention includes a protease variant having proteolytic activity and having improved stability relative to BPN′-v3, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acid substitutions selected from those listed in Table 12-6, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Each such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Table 12-7 provides the Performance Index (PI) values of BPN′ variants (generated as described in “Generation of BPN′ Variants from Five Different Plasmids”; see Table 11-4) for stain removal in a BMI microswatch assay in Detergent Composition lat 16° C. and pH 8. Assays were performed as described in Example 1 (BMI microswatch assay). The Performance Index of each variant was calculated relative to BPN′-v3: G97A-G128A-Y217Q. All mutants in this list have a PI cutoff equal or greater than 0.5 for at least one property tested.

TABLE 12-7 Performance Index of BPN′ Variants Sequence Relative to Det. Comp. 1, BPN′-v3 G97A- pH 8, 16° G128A-Y217Q Sequence Relative to BPN′ C., BMI PI S101N G97A-S101N-G128A-Y217Q 1.12 A137V G97A-G128A-A137V-Y217Q 1.12 N61P N61P-G97A-G128A-Y217Q 1.11 S130P G97A-G128A-S130P-Y217Q 1.09 Q103N G97A-Q103N-G128A-Y217Q 1.07 S63T S63T-G97A-G128A-Y217Q 1.03 G102A G97A-G102A-G128A-Y217Q 1.02 BPN′-v3 BPN′-v3 (G97A-G128A-Y217Q) 1.00 N109D-S248R G97A-N109D-G128A-Y217Q-S248R 0.96 S87R S87R-G97A-G128A-Y217Q 0.95 S188D G97A-G128A-S188D-Y217Q 0.95 S87D-S248R S87D-G97A-G128A-Y217Q-S248R 0.94 S188D-S248R G97A-G128A-S188D-S248R-Y217Q 0.93 S248D G97A-G128A-S248D-Y217Q 0.86 N109D-S188D- G97A-N109D-G128A-S188D- 0.83 S248R S248R-Y217Q N109D G97A-N109D-G128A-Y217Q 0.81 S87R-S248R S87R-G97A-G128A-Y217Q-S248R 0.79 N109D-S188R G97A-N109D-G128A-S188R- 0.77 Y217Q N76D N76D-G97A-G128A-Y217Q 0.75 S87D-N109D- S87D-G97A-N109D-G128A- 0.58 S188D-S248R S188D-S248R-Y217Q S87R-N109D- S87R-G97A-N109D-G128A- 0.55 S188D-S248R S188D-Y217Q-S248R S87R-S188R- S87R-G97A-G128A-S188R- 0.52 S248R Y217Q-S248R A187D G97A-G128A-A187D-Y217Q 0.48 N109D-S248D G97A-N109D-G128A-Y217Q- 0.47 S248D S87R-N109R- S87R-G97A-N109R-G128A- 0.39 S188R-S248R S188R-Y217Q-S248R F189D G97A-G128A-F189D-Y217Q 0.31 G100N G97A-G100N-G128A-Y217Q 0.28 S87R-N109D- S87R-G97A-N109D-G128A- 0.24 S188D S188D-Y217Q S87D-N109D- S87D-G97A-N109D-G128A- 0.12 S188D S188D-Y217Q S87R-S188D- S87R-G97A-G128A-S188D- 0.09 S248D S248D-Y217Q N62D N62D-G97A-G128A-Y217Q 0.09 S87D-N109D- S87D-G97A-N109D-G128A- 0.08 S188D-S248D S188D-Y217Q-S248D

The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acid substitutions selected from those listed in Table 12-7, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Each such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Table 12-8 provides the Performance index (PI) values of BPN′ variants (generated as described in “Generation of Combinatorial Libraries and Variants of BPN′-v3+578N” as described in Example 3) for stain removal using a BMI microswatch assay in Detergent Composition 1 at 16° C. and pH 8. Assays were performed as described in Example 1 (BMI microswatch assay). The Performance Index of each variant was calculated relative to BPN′-S78N-G97A-G128A-Y217Q. PI values less than 0.01 were modified and are indicated as “0.01” in bold italics.

TABLE 12-8 Performance Index Values of BPN′ Variants Sequence Det. Relative to Comp. BPN′-v3: 1 pH 8, Sequence G97A- 16° C., Variant Relative to BPN′ G128A-Y217Q BMI PI v3/S78N/L267V S78N-G97A-G128A- S78N-L267V 1.12 Y217Q-L267V v3/S78N/S161P S78N-G97A-G128A- S78N-S161P 1.05 Y217Q-S161P v3/S78N/I115V S78N-G97A-G128A- S78N-I115V 1.04 Y217Q-I115V v3/S78N/A273S S78N-G97A-G128A- S78N-A273S 1.03 Y217Q-A273S v3/S78N/G211T S78N-G97A-G128A- S78N-G211T 1.00 Y217Q-G211T V3 + S78N S78N-G97A-G128A- S78N 1.00 Y217Q v3/S78N/I111V S78N-G97A-G128A- S78N-I111V 0.98 Y217Q-I111V v3/S78N/V147L S78N-G97A-G128A- S78N-V147L 0.97 Y217Q-V147L v3/S78N/I108V S78N-G97A-G128A- S78N-I108V 0.97 Y217Q-I108V v3/S78N/S89Y S78N-G97A-G128A- S78N-S89Y 0.94 Y217Q-S89Y v3/S78N/A138T S78N-G97A-G128A- S78N-A138T 0.92 Y217Q-A138T v3/S78N/P172V S78N-G97A-G128A- S78N-P172V 0.74 Y217Q-P172V v3/S78N/Q59G S78N-G97A-G128A- S78N-Q59G 0.64 Y217Q-Q59G GcM96 G97A-G128A-Y217Q- P129T-V147Q- 0.57 P129T-V147Q-S159D- S159D-S161P- S161P-S183T-Q185T- S183T-Q185T- G211A-S224A G211A-S224A GcM91 G97A-G128A-Y217Q- Q059V-I108V- 0.55 Q059V-I108V-V147Q- V147Q-G211A- G211A-N252Q N252Q v3/S78N/Y167A S78N-G97A-G128A- S78N-Y167A 0.53 Y217Q-Y167A v3/S78N/A92G S78N-G97A-G128A- S78N-A92G 0.49 Y217Q-A92G v3/S78N/P129L S78N-G97A-G128A- S78N-P129L 0.48 Y217Q-P129L GcM92 G97A-G128A-Y217Q- N061A-S087E- 0.36 N061A-S087E-M124I- M124I-S161P- S161P-S224A S224A v3/S78N/N62Q S78N-G97A-G128A- S78N-N62Q 0.27 Y217Q-N62Q v3/S78N/V68A S78N-G97A-G128A- S78N-V68A 0.24 Y217Q-V68A GcM94 G97A-G128A-Y217Q- S063T-S101A- 0.12 S063T-S101A-L126V- L126V-S183T- S183T-T244N T244N v3/S78N/M124T S78N-G97A-G128A- S78N-M124T 0.05 Y217Q-M124T GcM95 G97A-G128A-Y217Q- P040L-S053G-

P040L-S053G-Q059V- Q059V-N061A- N061A-N062Q-S063T- N062Q-S063T- S087E-G100N S087E-G100N GcM93 G97A-G128A-Y217Q- N062Q-G100N-

N062Q-G100N-S125A- S125A-S159D- S159D-N240S N240S GcM100 G97A-G128A-Y217Q- V68A-G102A-

V68A-G102A-G211A- G211A-S125A S125A GcM90 G97A-G128A-Y217Q- S053G-V068A-

S053G-V068A-G102A- G102A-P129T- P129T-Q185T Q185T

The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one set of amino acid substitutions selected from those listed in Table 12-8, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Each such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Table 12-9 provides Performance index (PI) values of BPN′ single variants (constructed using PCR fusion as described in PCT App. No. PCT/US09/46156, filed Jun. 3, 2009, which is incorporated by reference herein for such description) for stain removal in a BMI microswatch assay in Detergent Composition 2 at 16° C. and pH 8 and for stability measured in Detergent Composition 3. PI values for specific activity by AAPF hydrolysis (PI specific AAPF) were determined. All assays were performed as described in Example 1. Performance index values were calculated relative to BPN wild type. PI values less than 0.01 are indicated as “0.01” in bold italics. “Det. Comp.” means Detergent Composition.

TABLE 12-9 Performance Index Values for BPN′ Single Variants BPN′ Det. Comp. 2 PI Specific Det. Comp. 3 Variant pH 8, 16° C., BMI PI AAPF Stability PI S182E 1.34 1.05 0.50 N109I 1.28 1.22 0.20 N117H 1.15 0.25 0.20 K237D 1.15 0.60 0.70 L257Q 1.14 0.94 0.80 P225N 1.13 1.02 0.70 S105H 1.11 1.07

S236I 1.10 0.58 0.90 L235H 1.10 0.65 0.70 S249E 1.07 0.72 0.30 N76E 1.07 0.76 0.20 S145N 1.06 1.16 1.10 N243D 1.05 1.03 0.90 R247N 1.04 1.04 0.50 E195N 1.04 1.05 0.40 A98K 1.03 0.75 0.70 S182N 0.99 1.14 0.90 S161H 0.97 1.07 0.90 G83H 0.95 0.72 0.60 G131D 0.95 1.11 1.30 T71C 0.93 1.00 1.30 K136Q 0.93 1.02 0.80 P40D 0.93 1.20 1.20 A187H 0.91 0.95 0.60 L250K 0.90 1.02 0.40 S9I 0.87 0.20

N76M 0.85 0.60 0.60 S132D 0.85 0.88 0.70 Q19F 0.83 0.47 0.30 E112H 0.83 1.02

S249P 0.80 1.02 0.50 S53D 0.78 0.29 0.10 V68E 0.78 0.59 1.70 D41I 0.72 1.15 0.90 K43H 0.70 0.28 0.10 V4H 0.66 0.37 0.60 A13Y 0.64 0.48

N62P 0.61 0.60 1.30 L196E 0.56 0.70 0.70 V44D 0.51 0.18

The invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and at least one substitution selected from those listed in Table 12-9, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence. Each such protease variant may be an isolated, recombinant, substantially pure, or non-naturally occurring protease variant. Also included are compositions, including cleaning compositions, comprising at least one such protease variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Example 13 Hand Dish Liquid Detergent Compositions

In this Example, various hand dish liquid detergent formulations are provided. The following hand dish liquid detergent compositions of the present invention are provided below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative aspects, other concentrations will find use, as determined by the formulator, based on their needs.

TABLE 13-1 Light-Duty Liquid Dishwashing Detergent Composition Composition 1 2 3 4 Linear Alkylbenzene Sulfonate (1) — — — Alkyl Ethoxy Sulfate (2) 18%  17% 17%  18%  Paraffin Sulfonate (C15) — — — — CAP = coco amido propyl Betaine — — 9% 5% Nonionic (3) — — 1% — Amine Oxide (4)  6% 5.5% — 4% Alkylpolyglucoside 4% Alcohol (5) — — 5% 7% Pura = polypropyleneglycol  1% 0.8% — — Citrate — — 0.3%  0.6%  Salt (6) 1.2%  1.0% — 0.5%  SCS = sodium cumene sulfonate — — 0.8%  — glycerol 15%  5% 3% — Na-lactate — — — 5% cationic polymer (7) 0.1%  0.1% 0.3%  0.2%  Protease of this invention    0.0075    0.0050    0.0025    0.030 Glycol distearate from Euperlan ® Cognis   0.4 0   0.4 0 Hydrogenated Castor Oil Thixcin ® Elementis 0   0.1 0   0.1 Mica (BASF Mearlin superfine) 0   0.05 0   0.05 Minors* Balance to 100% with water  pH 9 9 6 6 Optional Minors*: dyes, opacifier, perfumes, preservatives, hydrotropes, processing aids, and/or stabilizers. (1) Linear Alkylbenzene Sulfonate: LAS: C11.4 (2) Alkyl Ethoxy Sulfate: AExS: (3) Nonionic: AlkylEthoxylate (4) Di-methyl coco alkyl amine oxide (5) Alcohol: Ethanol (6) Salt: NaCl (7) cationically modified hydroxyethyl cellulose (Polyquaternium-10 - UCARE LR-400 ex Amerchol).

Example 14 Liquid & Granular Laundry Detergents

This Example provides various formulations for liquid laundry detergents. The following liquid laundry detergent formulations of the present invention are provided below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative aspects, other concentrations will find use, as determined by the formulator, based on their needs.

Liquid laundry detergent compositions suitable for front-loading automatic washing machines.

Composition (wt % of composition) Ingredient 1 2 3 4 5 6 7 8 Alkylbenzene sulfonic acid 7 11 4.5 1.2 1.5 12.5 5.2 4 Sodium C₁₂₋₁₄ alkyl ethoxy 3 2.3 3.5 4.5 4.5 7 18 1.8 2 sulfate C₁₄₋₁₅ alkyl 8-ethoxylate 5 8 2.5 2.6 4.5 4 3.7 2 C₁₂ alkyl dimethyl amine oxide — — 0.2 — — — — — C₁₂₋₁₄ alkyl hydroxyethyl dimethyl — — — 0.5 — — — — ammonium chloride C₁₂₋₁₈ Fatty acid 2.6 4 4 2.6 2.8 11 2.6 1.5 Citric acid 2.6 3 1.5 2 2.5 3.5 2.6 2 Cold Water Protease* 0.05 0.03 0.04 0.03 0.04 0.03 0.03 0.02 Amylase (Natalase ®) 0.1 0.2 0.15 — 0.05 0.5 0.1 0.2 Mannanase (Mannaway ®) 0.05 0.1 0.05 — — 0.1 0.04 — Random graft co-polymer¹ 1 0.2 1 0.4 0.5 2.7 0.3 1 A compound having the following 0.4 2 0.4 0.6 1.5 1.8 0.7 0.3 general structure: bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n), wherein n = from 20 to 30, and x = from 3 to 8, or sulphated or sulphonated variants thereof Ethoxylated Polyethylenimine² — — — — — 0.5 — — Amphiphilic alkoxylated grease 0.1 0.2 0.1 0.2 0.3 0.3 0.2 0.3 cleaning polymer³ Diethoxylated poly (1,2 propylene — — — — — — 0.3 — terephthalate short block soil release polymer. Diethylenetriaminepenta(methylenephosphonic) 0.2 0.3 — — 0.2 — 0.2 0.3 acid Hydroxyethane diphosphonic acid — — 0.45 — — 1.5 — 0.1 FWA 0.1 0.2 0.1 — — 0.2 0.05 0.1 Solvents (1,2 propanediol, 3 4 1.5 1.5 2 4.3 2 1.5 ethanol), stabilizers Hydrogenated castor oil derivative 0.4 0.4 0.3 0.1 0.3 — 0.4 0.5 structurant Boric acid 1.5 2.5 1.5 1.5 0.5 1.5 1.5 Na formate — — — 1 — — — — Reversible protease inhibitor⁴ — — 0.002 — — — — — Perfume 0.5 0.7 0.5 0.5 0.8 1.5 0.5 0.8 Perfume MicroCapsules slurry 0.2 0.3 0.7 0.2 0.05 0.4 0.9 0.7 (30% am) Ethoxylated thiophene Hueing 0.005 0.007 0.010 0.008 0.008 0.007 0.007 0.008 Dye⁵ Buffers (sodium hydroxide, To pH 8.2 Monoethanolamine) Water and minors (antifoam, To 100% aesthetics) Liquid laundry detergent compositions suitable for top-loading automatic washing machines.

Composition (wt % of composition) Ingredient 9 10 11 12 13 14 15 16 C₁₂₋₁₅ 20.1 15.1 20.0 15.1 13.7 16.7 10.0 9.9 Alkylethoxy(1.8)sulfate C_(11.8) Alkylbenzene sulfonate 2.7 2.0 1.0 2.0 5.5 5.6 3.0 3.9 C₁₆₋₁₇ Branched alkyl sulfate 6.5 4.9 4.9 3.0 9.0 2.0 C₁₂₋₁₄ Alkyl-9-ethoxylate 0.8 0.8 0.8 0.8 8.0 1.5 0.3 11.5 C₁₂ dimethylamine oxide 0.9 Citric acid 3.8 3.8 3.8 3.8 3.5 3.5 2.0 2.1 C₁₂₋₁₈ fatty acid 2.0 1.5 2.0 1.5 4.5 2.3 0.9 Cold Water Protease* 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 Amylase (Natalase ®) 0.7 0.3 0.6 0.3 0.6 0.4 Amylase (Termamyl Ultra ®) 1.1 Mannanase (Mannaway ®) 0.1 0.1 Pectate Lyase (Pectawash ®) 0.1 0.2 Borax 3.0 3.0 2.0 3.0 3.0 3.3 Na & Ca formate 0.2 0.2 0.2 0.2 0.7 A compound having the 1.6 1.6 3.0 1.6 2.0 1.6 1.3 1.2 following general structure: bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)- bis((C₂H₅O)(C₂H₄O)n), wherein n = from 20 to 30, and x = from 3 to 8, or sulphated or sulphonated variants thereof Random graft co-polymer¹ 0.4 0.2 1.0 0.5 0.6 1.0 0.8 1.0 Diethylene triamine 0.4 0.4 0.4 0.4 0.2 0.3 0.8 pentaacetic acid Tinopal AMS-GX 0.2 0.2 0.2 0.2 0.2 0.3 0.1 Tinopal CBS-X 0.1 0.2 Amphiphilic alkoxylated 1.0 1.3 1.3 1.4 1.0 1.1 1.0 1.0 grease cleaning polymer³ Texcare 240N (Clariant) 1.0 Ethanol 2.6 2.6 2.6 2.6 1.8 3.0 1.3 Propylene Glycol 4.6 4.6 4.6 4.6 3.0 4.0 2.5 Diethylene glycol 3.0 3.0 3.0 3.0 3.0 2.7 3.6 Polyethylene glycol 0.2 0.2 0.2 0.2 0.1 0.3 0.1 1.4 Monoethanolamine 2.7 2.7 2.7 2.7 4.7 3.3 1.7 0.4 Triethanolamine 0.9 NaOH to pH to pH to pH to pH to pH to pH to pH to pH 8.3 8.3 8.3 8.3 8.3 8.3 8.3 8.5 Suds suppressor Dye 0.01 0.01 0.01 0.01 0.01 0.01 0.0 Perfume 0.5 0.5 0.5 0.5 0.7 0.7 0.8 0.6 Perfume MicroCapsules 0.2 0.5 0.2 0.3 0.1 0.3 0.9 1.0 slurry (30% am) Ethoxylated thiophene 0.003 0.002 0.002 0.005 0.002 0.004 0.004 0.003 Hueing Dye⁵ Water balance balance balance balance balance balance balance balance

Granular detergent compositions produced in accordance with the invention suitable for laundering fabrics.

17 18 19 20 21 22 Linear alkylbenzenesulfonate 15  12  20  10  12  13  with aliphatic carbon chain length C₁₁-C₁₂ Other surfactants 1.6 1.2 1.9 3.2 0.5 1.2 Phosphate builder(s) 2  3  4  Zeolite 1  1  4  1  Silicate 4  5  2  3  3  5  Sodium Carbonate 2  5  5  4  0  3  Polyacrylate (MW 4500) 1  0.6 1  1  1.5 1 Carboxymethyl cellulose 1  — 0.3 — 1.1 — (Finnfix BDA ex CPKelco) Celluclean ®  0.23  0.17 0.5 0.2 0.2 0.6 Cold Water Protease*  0.23  0.17 0.5 0.2 0.2 0.6 Stainzyme Plus ®  0.23  0.17 0.5 0.2 0.2 0.6 Fluorescent Brightener(s)  0.16  0.06  0.16  0.18  0.16  0.16 Diethylenetriamine pentaacetic 0.6 0.6  0.25 0.6 0.6 acid or Ethylene diamine tetraacetic acid MgSO₄ 1  1  1  0.5 1 1 Bleach(es) and Bleach  6.88  6.12  2.09  1.17  4.66 activator(s) Ethoxylated thiophene Hueing  0.002  0.001  0.003  0.003 — — Dye⁵ Direct Violet 9 ex Ciba Specialty   0.0006   0.0004   0.0006 Chemicals Sulfate/Citric Acid/Sodium Balance to 100% Bicarbonate/Moisture/perfume ¹Random graft copolymer is a polyvinyl acetate grafted polyethylene oxide copolymer having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is about 6000 and the weight ratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60 and no than 1 grafting point per 50 ethylene oxide units. ²Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH. ³Amphiphilic alkoxylated grease cleaning polymer is a polyethylenimine (MW = 600) with 24 ethoxylate groups per —NH and 16 propoxylate groups per —NH ⁴Reversible Protease inhibitor of structure:

⁵Ethoxylated thiophene Hueing Dye is a described in U.S. Pat. No. 7,208,459 B2. *Remark: all enzyme levels expressed as % enzyme raw material, except for cold water protease (of this invention) which is expressed as % of active protein added to the product. .

Example 15 Unit Dose Compositions

This Example provides various formulations for unit dose laundry detergents. Such unit dose formulations can comprise one or multiple compartments.

The following unit dose laundry detergent formulations of the present invention are provided below.

TABLE 15-1 Unit Dose Compositions Ingredients 1 2 3 4 5 Alkylbenzene sulfonic acid C 11-13, 14.5 14.5 14.5 14.5 14.5 23.5% 2-phenyl isomer C₁₂₋₁₄ alkyl ethoxy 3 sulfate 7.5 7.5 7.5 7.5 7.5 C₁₂₋₁₄ alkyl 7-ethoxylate 13.0 13.0 13.0 13.0 13.0 Citric Acid 0.6 0.6 0.6 0.6 0.6 Fatty Acid 14.8 14.8 14.8 14.8 14.8 Enzymes (as % raw material not 1.7 1.7 1.7 1.7 1.7 active) Protease of this invention (as % 0.05 0.1 0.02 0.03 0.03 active) Ethoxylated Polyethylenimine¹ 4.0 4.0 4.0 4.0 4.0 Series 1 GG36 protease (as % active) 0.02 0 0.01 0.02 0.03 Hydroxyethane diphosphonic acid 1.2 1.2 1.2 1.2 1.2 Brightener 0.3 0.3 0.3 0.3 0.3 P-diol 15.8 13.8 13.8 13.8 13.8 Glycerol 6.1 6.1 6.1 6.1 6.1 MEA 8.0 8.0 8.0 8.0 8.0 TIPA — — 2.0 — — TEA — 2.0 — — — Cumene sulphonate — — — — 2.0 cyclohexyl dimethanol — — — 2.0 — Water 10 10 10 10 10 Structurant 0.14 0.14 0.14 0.14 0.14 Perfume 1.9 1.9 1.9 1.9 1.9 Buffers (monoethanolamine) To pH 8.0 Solvents (1,2 propanediol, ethanol) To 100% ¹Polyethylenimine (MW = 600) with 20 ethoxylate groups per —NH.

Multiple compartment unit dose laundry detergent formulations of the present invention are provided below. In these examples the unit dose has three compartments, but similar compositions can be made with two, four or five compartments. The film used to encapsulate the compartments is polyvinyl alcohol.

TABLE 15-2 Multiple Compartment Unit Dose Detergent Compositions Base Composition 1 Ingredients % Glycerol (min 99) 5.3 1,2-propanediol 10 Citric Acid 0.5 Monoethanolamine 10 Caustic soda — Dequest 2010 1.1 Potassium sulfite 0.2 Nonionic Marlipal C24EO7 20.1 HLAS (surfactant) 24.6 Optical brightener FWA49 0.2 C12-15 Fatty acid 16.4 Polymer Lutensit Z96 2.9 Polyethyleneimine ethoxylate 1.1 PEI600 E20 MgCl2 0.2 Solvents (1,2 propanediol, ethanol) To 100% Multi-compartment formulations Composition 1 2 Compartment A B C A B C Volume 40 ml 5 ml 5 ml 40 ml 5 ml 5 ml of each compartment Active material in Wt. % Perfume 1.6 1.6 1.6 1.6 1.6 1.6 Dyes <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 TiO2 0.1 — — — 0.1 — Sodium Sulfite 0.4 0.4 0.4 0.3 0.3 0.3 Acusol 305, 1.2 2 — — Rohm&Haas Hydrogenated 0.14 0.14 0.14 0.14 0.14 0.14 castor oil Base Add to Add to Add to Add to Add to Add to Composition 1 100% 100% 100% 100% 100% 100%

Example 16 Cleaning Performance of BPN′-v36 Polypeptide Variants

BPN′-v36 polypeptide variants comprising two amino acid substitutions were constructed by standard PCR fusion using the BPN′-v36 variant as a backbone or parent sequence. For this purpose, two or three partially overlapping fragments were amplified by mutagenic primers prepared such that the primer encoded a desired substitution. PCR amplification reactions were carried out as described in Example 7 of Part I supra. The following BPN′-v36 double mutant variants (i.e., BPN′-v36 with the following two amino acid substitution) were constructed: Q019R-N025D, A001Y-Q275R, V004A-S249N, V004E-S260P, V004A-T55A, Y006F-S249C, Y006D-T55A, V008L-Q275R, Q010R-Q275K, L016Q-Q217H, H017R-T158A, S183D-Q206R, P210S-N212D, S018Y-V203A, S018K-V203I, Y021H-D259G, Y021H-D259R, K027R-N269D, K027R-N269T, S037P-S260F, S037T-S260P, D041E-N077D, D041G-N077E, G166V-S183T, N252S-L257H, V044A-Q206H, V044A-Q206K, V044A-Q206R, N076T-N212D, N076P-N212S, N077D-N252D, N077D-N252T, K141I-S248N, T158I-D259N, T158A-D259P, S161E-Q185H, K237M-H238R, G160A-D259G, G160R-D259V, G215R-D259R, G215D-D259V, N061D-Q206R, N061L-Q206H, S009L-N218S, S161E-S260T, Q019A-N109S, T022S-G166V, Y021H-N252H, P129S-K136R, T022S-T242S, N025K-H238R, N025D-Q185R, S037G-Q275H, K043R-N076S, K043N-Q217R, K043N-S163T, T055A-V147A, N061K-N252K, N062Y-G097D, Y021H-V084E, Y021H-S037E, N062Y-T244A, K027E-Y091F, A074S-P129Q, S249R-Q275R, I079V-Q217H, A098T-T158A, K027R-D120H, Q019R-Q185R, G131S-K265N, A133V-D259N, A144H-T244A, I035V-K043N, G160R-T244A, S161P-T253A, S163T-Q245L, K170R-D259G, S183T-S249R, N184Y-Y262N, V198L-D259G, A200T-H226L, Q206R-S260P, G211V-T244A, Q217R-T244A, L75I-N76D, S260P-Q275L, S260P-Q275R, Y262N-Q275R, V004A-Y006F, H017L-Q019A, N025D-V026A, N118G-V121A, V072F-L075I, S183T-R186K, V203A-Q217R, and S249R-Y262H. The cleaning performance of these variants was tested in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C. and egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C. as described in Example 1 of Part I. Results are provided below.

The following BPN′ protease variants were determined to have a PI value equal to or greater than 0.9 and equal or less than 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v3, BPN′-v36, S183T-S249R, N61D-Q206R, Y262N-Q275R, K43R-N76S, S183T-S249R, K170R-D259G, Y6F-S249C, Q19A-N109S, H17L-Q19A, Q19R-Q185R, S18Y-V203A, N61D-Q206R, S161E-S260T, S18K-V203I, V4A-T55A, N252S-L257H, S249R-Y262H, N61L-Q206H, N184Y-Y262N, Q19R-N25D, S249R-Y262H, A74S-P129Q, H17L-Q19A, K27R-D120H, V4A-T55A, Y21H-N252H, K27R-N269D, K27R-N269D, A98T-T158A, I79V-Q217H, S9L-N218S, V4A-Y6F, S161P-T253A, V203A-Q217R, T22S-T242S, N76P-N212S, K170R-D259G, S37T-S260P, T55A-V147A, Q19R-Q185R, V4A-Y6F, Q19A-N109S, Y262N-Q275R, G160R-T244A, Q19R-N25D, N25D-Q185R, A98T-T158A, N61L-Q206H, G211V-T244A, S9L-N218S, A144H-T244A, A144H-T244A, S18Y-V203A, Y21H-N252H, A74S-P129Q, A1Y-Q275R, V198L-D259G, T55A-V147A, K141I-S248N, S183T-R186K, S37T-S260P, K27R-D120H, T22S-T242S, S161E-Q185H, P129S-K136R, G211V-T244A, N76P-N212S, K43N-S163T, S37G-Q275H, S161P-T253A, Y6F-S249C, N184Y-Y262N, N252S-L257H, G160R-T244A, S37G-Q275H, P129S-K136R, N62Y-T244A, and S260P-Q275R, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2) and a greater PI value than BPN′ in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), a PI value of equal to or greater than 0.9 and equal to or less than 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution comprising at least one substitution selected from the group of X1Y, X4A, X6F, X9L, X17L, X18K/Y, X19A/R, X21H, X22S, X25D, X27R, X37G/T, X43N/R, X55A, X61D/L, X62Y, X74S, X76P/S, X79V, X98T, X109S, X120H, X129Q/S, X136R, X141I, X144H, X147A, X158A, X160R, X161E/P, X163T, X170R, X183T, X184Y, X185H/R, X186K, X198L, X203A/I, X206H/R, X211V, X212S, X217H/R, X218S, X242S, X244A, X248N, X249C/R, X252H/S, X253A, X257H, X259G, X260P/T, X262H/N, X269D, and X275H/R, and optionally at least one substitution selected from the group of A1Y, V4A, Y6F, S9L, H17L, S18K/Y, Q19A/R, Y21H, T22S, N25D, K27R, S37G/T, K43N/R, T55A, N61D/L, N62Y, A74S, N76P/S, I79V, A98T, N109S, D120H, P129Q/S, K136R, K141I, A144H, V147A, T158A, G160R, S161E/P, S163T, K170R, S183T, N184Y, Q185H/R, R186K, V198L, V203A/I, Q206H/R, G211V, N212S, Q217H/R, N218S, T242S, T244A, S248N, S249C/R, N252H/S, T253A, L257H, D259G, S260P/T, Y262H/N, N269D, and Q275H/R, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′ subtilisin protease variants were determined to have a PI value equal or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of Y21H-D259G, Y21H-D259G, A133V-D259N, I79V-Q217H, S18K-V203I, T158A-D259P, N61K-N252K, K43N-Q217R, T158A-D259P, Q206R-S260P, A133V-D259N, V198L-D259G, N61K-N252K, S161E-S260T, G160A-D259G, K43N-Q217R, A1Y-Q275R, A200T-H226L, Q217R-T244A, S260P-Q275R, Q206R-S260P, T158I-D259N, Q217R-T244A, L75I-N76D, S161E-Q185H, Y21H-S37E, S249R-Q275R, G160A-D259G, T158I-D259N, Y21H-S37E, N76T-N212D, S260P-Q275L, G131S-K265N, V4A-S249N, N25D-Q185R, K43R-N76S, S183D-Q206R, Q10R-Q275K, K43N-S163T, Q10R-Q275K, N25D-V26A, G131S-K265N, S260P-Q275L, K141I-S248N, L16Q-Q217H, S249R-Q275R, K27R-N269T, P210S-N212D, L75I-N76D, S183D-Q206R, N118G-V121A, G215D-D259V, N76T-N212D, V4A-S249N, K27R-N269T, N62Y-G97D, V4E-S260P, G215D-D259V, K27E-Y91F, Y6D-T55A, N77D-N252T, V4E-S260P, Y6D-T55A, N77D-N252T, N25K-H238R, V44A-Q206H, L16Q-Q217H, S37P-S260F, V44A-Q206R, V44A-Q206H, V44A-Q206R, and S37P-S260F, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, having enhanced proteolytic activity greater than BPN′, and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of Y21H-D259G, S183T-S249R, N61D-Q206R, Y262N-Q275R, Y021H-D259G, K043R-N076S, S183T-S249R, A133V-D259N, and I079V-Q217H, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′ in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution comprising at least one substitution selected from the group of X021H, X043R, X061D, X076S, X079V, X133V, X183T, X206R, X217H, X249R, X259G/N, X262N, and X275R, and optionally at least one substitution selected from the group of Y021H, K043R, N061D, N076S, I079V, A133V, S183T, Q206R, Q217H, S249R, D259G/N, Y262N, and Q275R, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than or equal to 1.0 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′-v3, BPN′-v36, K170R-D259G, S18K-V203I, Y6F-S249C, Q19A-N109S, H17L-Q19A, Q19R-Q185R, S18Y-V203A, N61D-Q206R, S161E-S260T, S18K-V203I, V4A-T55A, N252S-L257H, S249R-Y262H, N61L-Q206H, N184Y-Y262N, Q19R-N25D, S249R-Y262H, A74S-P129Q, T158A-D259P, H17L-Q19A, K27R-D120H, V4A-T55A, N61K-N252K, Y21H-N252H, K27R-N269D, K43N-Q217R, T158A-D259P, Q206R-S260P, K27R-N269D, A98T-T158A, I79V-Q217H, S9L-N218S, V4A-Y6F, S161P-T253A, V203A-Q217R, T22S-T242S, N76P-N212S, A133V-D259N, K170R-D259G, S37T-S260P, T55A-V147A, V198L-D259G, Q19R-Q185R, V4A-Y6F, Q19A-N109S, Y262N-Q275R, G160R-T244A, Q19R-N25D, N25D-Q185R, N61K-N252K, S161E-S260T, A98T-T158A, N61L-Q206H, G211V-T244A, S9L-N218S, A144H-T244A, A144H-T244A, S18Y-V203A, Y21H-N252H, A74S-P129Q, G160A-D259G, K43N-Q217R, A1Y-Q275R, A1Y-Q275R, A200T-H226L, Q217R-T244A, S260P-Q275R, V198L-D259G, T55A-V147A, Q206R-S260P, K141I-S248N, S183T-R186K, T158I-D259N, S37T-S260P, K27R-D120H, T22S-T242S, Q217R-T244A, S161E-Q185H, P129S-K136R, G211V-T244A, N76P-N212S, L75I-N76D, S161E-Q185H, Y21H-S37E, S249R-Q275R, G160A-D259G, K43N-S163T, T158I-D259N, Y21H-S37E, S37G-Q275H, S161P-T253A, N76T-N212D, S260P-Q275L, Y6F-S249C, N184Y-Y262N, G131S-K265N, V4A-S249N, N25D-Q185R, N252S-L257H, K43R-N76S, S183D-Q206R, G160R-T244A, Q10R-Q275K, S37G-Q275H, K43N-S163T, Q10R-Q275K, N25D-V26A, P129S-K136R, G131S-K265N, S260P-Q275L, K141I-S248N, T22S-G166V, N62Y-T244A, L16Q-Q217H, S249R-Q275R, S260P-Q275R, K27R-N269T, P210S-N212D, L75I-N76D, S183D-Q206R, N118G-V121A, G215D-D259V, N76T-N212D, V4A-S249N, K27R-N269T, G166V-S183T, N62Y-G97D, V4E-S260P, G215D-D259V, K27E-Y91F, Y21H-D259R, Y6D-T55A, N77D-N252T, V4E-S260P, Y6D-T55A, N77D-N252T, Y21H-D259R, N25K-H238R, N77D-N252D, V44A-Q206H, L16Q-Q217H, V72F-L75I, S37P-S260F, V72F-L75I, N77D-N252D, V44A-Q206R, S163T-Q245L, V44A-Q206H, V44A-Q206R, S37P-S260F, G215R-D259R, V44A-Q206K, and V44A-Q206K, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and equal to or less than 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Example 17 Cleaning Performance of Additional BPN′-v36 Polypeptide Variants

The following BPN′-v36 variants were synthesized at DNA2.0 (Menlo Park, Calif.) using the pHPLT-BPN′-v36 plasmid containing the BPN′ expression cassette served as template DNA (parent plasmid) for cloning: N109G-A128S-S224A, N109G-A128S-S224A-N243V, A88T-N109G-A116T-A128S-S224A-N243V, N61G-N109G-A128S-S224A, N61G-N109G-A128S-S224A-N243V, N61G-A88T-N109G-A116T-A128S-S224A-N243V, N109Q-A128S-S224A,

N109Q-A128S-S224A-N243V, A88T-N109Q-A116T-A128S-S224A-N243V, N109S-A128S-S224A, N109S-A128S-S224A-N243V, A88T-N109S-A116T-A128S-S224A-N243V, N109M-A128S-S224A, N109M-A128S-S224A-N243V, A88T-N109M-A116T-A128S-S224A-N243V, N109G-A114S-A128S, N109G-A114S-A128S-N243V, A88T-N109G-A114S-A116T-A128S-N243V, N109G-A114S-A128S-S224A, N109G-A114S-A128S-S224A-N243V, N109G-A128S-S183V, N109G-A128S-S183L, N109G-A128S-S183L-S224A, N109G-A114S-A128S-S183L-S224A, A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N76D-N109G-A128S-S224A, N101Q-N109Q-A128S-S224A-N243V, N101Q-N109Q-A128S-P129S-S130T-S224A-N243V, N109G-A128S-P129S-S130T-S224A-N243V, S33T-A128S-N218S, S33T-N109G-A128S-N218S-N243V, S33T-N61G-N109G-A128S-N218S-N243V, S33T-N109G-A128S-G169A-N218S-N243V, S33T-S63G-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V, S33T-S63G-N109G-A128S-G169A-N218S-N243V, I31L-S33T-S63G-N109G-A128S-G169A-N218S-N243V, S33T-N61G-S63G-N109G-A128S-N218S-N243V, S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V, S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V, S33T-N61P-S63G-N109G-A128S-G131H-G169A-N218S-N243V, S33T-N109G-A128S-N218S-S224A-N243V, S63G-N109Q-A128S-S224A-N243V, S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-S224A-N243V, N61P-S63G-N109Q-A128S-G131H-S224A-N243V, A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, I31L-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q, S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V, S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q, A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q, N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q, and A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q.

The variants were grown for protein expression as described in Example 11 of Part I. These variants were tested for their performance in the BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., and the AAPF assay as described in Example 1 of Part I. Results are provided below.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of N61P-S63G-N109Q-A128S-S224A-N243V, A88T-N109G-A114S-A116T-A128S-N243V, A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A128S-S183V, N109G-A128S-N243V-K256R, N109M-A128S-S224A, A88T-N109S-A116T-A128S-S224A-N243V, N109Q-A128S-S224A-N243V, A88T-N109M-A116T-A128S-S224A-N243V, N109S-A128S-S224A-N243V, A88T-N109G-A116T-N243V, N101Q-N109Q-A128S-S224A-N243V, N109G-A116T-N243V-K256R, N109G-A128S-P129S-S130T-S224A-N243V, and A88T-N109Q-A116T-A128S-S224A-N243V, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′-v36 and/or a PI value of equal to or greater than 1.0 compared to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution comprising at least one substitution selected from the group of X61G/P/S, X63G, X88T, X101Q, X109G/M/Q/S, X114S, X116T, X128S, X129S, X130T, X158S, X183L/V, X224A, X243V, X248A, and X256R, and optionally at least one substitution selected from the group of N61G/P/S, S63G, A88T, N101Q, N109G/M/Q/S, A114S, A116T, A128S, P129S, S130T, T158S, S183L/V, S224A, N243V, S248A, and K256R, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.9 and equal to or less than 1.0 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of G24S-G53S-N78S-G97A-N101S-A128S, G24S-G53S-N78S-G97A-N101S, BPN′-v36, S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V, S33T-N61G-S63G-N109G-A128S-N218S-N243V, S33T-S63G-N109G-A128S-N218S-N243V, S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q, S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V, S33T-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V-S248N-K256R, S33T-N61G-N109G-A128S-N218S-N243V, S33T-A128S-N218S, A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q, N61P-S63G-N109Q-A128S-G131H-S224A-N243V, S63G-N109Q-A128S-G131H-S224A-N243V, N109G-A114S-A128S, N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A, N109G-A114S-A128S-S224A-N243V, A88T-N109G-A116T-A128S-S224A-N243V, N61G-A88T-N109G-A116T-A128S-S224A-N243V, N109G-A114S-A128S-N243V, N109G-A128S-S224A-N243V, N109G-A128S-S224A, N109G-A128S-S183L-S224A, N61G-N109G-A128S-S224A, N109G-A128S-S183L, S33T-N76D, N109S-A128S-S224A, N101Q-N109Q-A128S-P129S-S130T-S224A-N243V, S63G-N109Q-A128S-S224A-N243V, N109M-A128S-S224A-N243V, S63G-N109G, N109G-K256R, S63G-N76D, S33T-N109G-A128S-G169A-N218S-N243V, and S33T-N109G-A128S-N218S-S224A-N243V, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity, enhanced proteolytic activity compared to BPN′, and/or a PI value equal to or greater than 0.9 and less or equal to 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of 131L-S33T-S63G-N109G-A128S-G169A-N218S-N243V, A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q, S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V, S33T-S63G-N109G-A128S-G169A-N218S-N243V, S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q, S33T-N76D-A128S-N218S, N76D-N109G-A128S-S224A, and S33T-N61P-S63G-N109G-A128S-G131H-G169A-N218S-N243V, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V, S33T-N61G-S63G-N109G-A128S-N218S-N243V, S33T-S63G-N109G-A128S-N218S-N243V, N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q, S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V, S33T-N109G-A128S-N218S-N243V, S33T-A128S-N218S, A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q, S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-S224A-N243V, A88T-N109G-A114S-A116T-A128S-N243V, A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A114S-A128S, N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A, N109G-A114S-A128S-S224A-N243V, A88T-N109G-A116T-A128S-S224A-N243V, N61G-A88T-N109G-A116T-A128S-S224A-N243V, N109G-A128S-S183V, N109G-A114S-A128S-N243V, N109G-A128S-N243V-S248A, N109G-A128S-S224A-N243V, N109G-A128S-N243V-K256R, N109G-A128S-S224A, N109G-A128S-S183L-S224A, N61G-N109G-A128S-S224A, N109M-A128S-S224A, A88T-N109S-A116T-A128S-S224A-N243V, N109M-A128S-S224A-N243V, S63G-A128S, A88T-N109G-A116T-N243V, N101Q-N109Q-A128S-S224A-N243V, N109G-A116T-N243V-K256R, N109G-A116T, S63G-N109G, A88T-N109G, N109G-K256R, N61G-N109G-N243V, S33T-N109G-A128S-G169A-N218S-N243V, S33T-N109G-A128S-N218S-S224A-N243V, N109G-A128S-P129S-S130T-S224A-N243V, and A88T-N109Q-A116T-A128S-S224A-N243V, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′-v36 and/or a PI value of greater than 1.0 compared to BPN′-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C., the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution comprising at least one substitution selected from the group of X1G, X33T, X55P, X61G/P/S, X63G, X76D, X88T, X101Q, X109G/M/Q/S, X114S, X116T, X128S, X129S, X130T, X131H, X158S, X169A, X183L/V, X218S, X224A, X243V, X248A/N, X249Q, X256R, and optionally at least one substitution selected from the group of A1G, S33T, T55P, N61G/P/S, S63G, N76D, A88T, N101Q, N109G/M/Q/S, A114S, A116T, A128S, P129S, S130T, G131H, T158S, G169A, S183L/V, N218S, S224A, N243V, S248A/N, S249Q, K256R, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variant was determined to have a PI value equal to or greater than 0.5 and equal to or less than 1.0 relative to BPN′-v36 in an egg BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising amino acid substitutions selected from the group consisting of substitutions S33T-N76D-A128S-N218S, N76D-N109G-A128S-S224A, and S063G-N76D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and equal to or less than 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising amino acid substitutions selected from the group consisting of S33T-N76D-A128S-N218S, N76D-N109G-A128S-S224A, and S063G-N076D, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN′-v36 in an AAPF proteolytic assay: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of G24S-G53S-N78S-G97A-N101S-A128S, I31L-S33T-S63G-N109G-A128S-G169A-N218S-N243V, A1G-I31L-S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-G169A-S224A-N243V-S249Q, S33T-N61G-S63G-N109G-A128S-G131H-G169A-N218S-N243V, S33T-S63G-N109G-A128S-G169A-N218S-N243V, S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V, S33T-T55P-N61P-S63G-A88T-N109G-A116T-A128S-G131H-S224A-N243V-S249Q, S33T-N61G-S63G-N109G-A128S-N218S-N243V, S33T-S63G-N109G-A128S-N218S-N243V, S33T-T55P-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-G131H-G169A-S224A-N243V-S249Q, S33T-N61G-A88T-N109G-A116T-A128S-N218S-N243V, S33T-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V, S33T-N76D-N109G-A128S-N218S-N243V-S248N-K256R, S33T-N61G-N109G-A128S-N218S-N243V, S33T-N76D-A128S-N218S, S33T-A128S-N218S, A1G-N61P-S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-G131H-S224A-N243V-S249Q, N61P-S63G-N109Q-A128S-G131H-S224A-N243V, S63G-N109Q-A128S-G131H-S224A-N243V, N61P-S63G-N109Q-A128S-S224A-N243V, A88T-N109G-A114S-A116T-A128S-N243V, A88T-N109G-A114S-A116T-A128S-S183L-S224A-N243V, N109G-A114S-A128S, N109G-A114S-A128S-S183L-S224A, N109G-A114S-A128S-S224A, N109G-A114S-A128S-S224A-N243V, A88T-N109G-A116T-A128S-S224A-N243V, N61G-A88T-N109G-A116T-A128S-S224A-N243V, N109G-A128S-S183V, N109G-A114S-A128S-N243V, N109G-A128S-N243V-S248A, N109G-A128S-S224A-N243V, N109G-A128S-N243V-K256R, N109G-A128S-S224A, N109G-A128S-S183L-S224A, N61G-N109G-A128S-S224A, N76D-N109G-A128S-S224A, N109M-A128S-S224A, N109G-A128S-S183L, S33T-N76D, A88T-N109S-A116T-A128S-S224A-N243V, N109Q-A128S-S224A-N243V, N109S-A128S-S224A, A88T-N109M-A116T-A128S-S224A-N243V, N101Q-N109Q-A128S-P129S-S130T-S224A-N243V, S63G-N109Q-A128S-S224A-N243V, N109M-A128S-S224A-N243V, S63G-A128S, N109S-A128S-S224A-N243V, A88T-N109G-A116T-N243V, N61S-N109G-N243V, N101Q-N109Q-A128S-S224A-N243V, N109G-A116T-N243V-K256R, A88T-N109G-A116T-T158S-N243V-K256R, N109G-A116T, S63G-N109G, A88T-N109G, N109G-K256R, N61G-N109G-N243V, S33T-N61P-S63G-N109G-A128S-G131H-G169A-N218S-N243V, S33T-N109G-A128S-G169A-N218S-N243V, S33T-N109G-A128S-N218S-S224A-N243V, N109G-A128S-P129S-S130T-S224A-N243V, and A88T-N109Q-A116T-A128S-S224A-N243V, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′, BPN′-v3, and BPN′-v36, and a greater PI value than BPN′, BPN′-v3 and BPN′-v36 in this assay. The invention includes a protease variant having enhanced proteolytic activity compared to BPN′ (SEQ ID NO:2), enhanced proteolytic activity compared to BPN′, BPN′-v3, and BPN′-v36, a PI value of greater than 1.0 to about 5 relative to BPN′-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Also provided is a subtilisin protease variant having enhanced proteolytic activity compared to BPN′-v36 and/or a PI value of greater than 1.0 compared to BPN′-v36 in An AAPF proteolytic assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution comprising at least one substitution selected from the group of X1G, X31L, X33T, X55P, X61G/P/S, X63G, X76D, X88T, X101Q, X109G/M/Q/S, X114S, X116T, X128S, X129S, X130T, X131H, X158S, X169A, X183L/V, X218S, X224A, X243V, X248A/N, X249Q, and X256R, and optionally at least one substitution selected from the group of A1G, I31L, S33T, T55P, N61G/P/S, S63G, N76D, A88T, N101Q, N109G/M/Q/S, A114S, A116T, A128S, P129S, S130T, G131H, T158S, G169A, S183L/V, N218S, S224A, N243V, S248A/N, S249Q, and K256R, wherein amino acid positions of the variant are numbered by correspondence with positions of the sequence of SEQ ID NO:2. Such variants have enhanced proteolytic activity compared to the BPN′ (SEQ ID NO:2) BPN′-v3, and BPN′-v36 and a PI value greater than that of BPN′, BPN′-v3, and BPN′-v36 in this assay. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variant was determined to have a PI value equal to or greater than 0.5 and equal to or less than 1.0 relative to BPN′-v36 in an AAPF proteolytic assay: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising amino acid substitutions G24S-G53S-N78S-G97A-N101S or S063G-N76D, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and equal to or less than 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising substitutions G24S-G53S-N78S-G97A-N101S or S063G-N76D, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Example 18 Cleaning Performance of Polypeptide BPN′-v36 Polypeptides Variants from Combinatorial Libraries Based on BPN′-v36 Polypeptide

Two separate combinatorial libraries (AJ1 and AJ2) were synthesized by DNA2.0 (Menlo Park, Calif.) and were delivered as individual ligation reactions. The pHPLT-BPN′-v36 plasmid (FIG. 4) containing the BPN′ expression cassette served as template DNA (parent plasmid) for library construction. A list of the possible amino acid positions and substitutions for each library is shown in Table 23-1. The ligation reactions for each library were used to transform B. subtilis, and the library variants were grown up for protein expression as described in Example 11The variants were tested for performance in the BMI microswatch cleaning assay in Detergent Composition 4 (from Table 1-3) at pH 8 and 16° C. as described in Example 1.

TABLE 18-1 Possible Substitutions for Combinatorial Libraries AJ1 and AJ2 AJ1 AJ2 Possible Possible Position Substitutions Position Substitutions S33 G, S E54 E, Q D60 D, G D99 D, N N62 N, L, S D120 D, N S63 S, R, L, N, G D140 D, N S125 S, A E156 E, Q Q217 Q, R, E, L, G D197 D, N M222 M, L, S K12 K, T K27 K, S K43 K, T K141 K, Y K213 K, Q K237 K, A K256 K, Q

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less or equal to 1.0 relative to BPN′-v36 in the BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN′v36, BPN′v3 (G024S-G053S-N078S-G097A-N101S), BPN′v3 (G024S-G053S-N078S-G097A-N101S, BPN′ v12 (G024S-G053S-N078S-G097A-N101S-A128S), N062L, N062L-S063G, N062S, N062S-S063G-Q217L, N062S-S063L-Q217L, N062S-S063N, N062S-S063R, Q217E, S063G, S063G-Q217L, S063G-Q217L-M222S, S063L-Q217L, S063N, S063N-Q217L, D099N-K141Y-K213Q, D099N-K141Y-K256Q, K043T, K043T-K141Y-E156Q, N062L-Q217E, N062L-Q217L, N062L-S063G-Q217E, N062L-S063L, N062L-S063N-Q217L, N062S-Q217L, N062S-S063G, N062S-S063L, N062S-S063N-Q217L, N062S-S063R-Q217E, Q217L, S063G-Q217E, S063N-Q217E, S063R, S063R-Q217E, S063R-Q217L,), D099N-K141Y-K213Q, D099N-K141Y-K256Q, K043T, K043T-K141Y-E156Q, N062L-Q217E, N062L-Q217L, N062L-S063G-Q217E, N062L-S063L, N062S-Q217L, N062S-S063G, N062S-S063L, N062S-S063N-Q217L, Q217L, S063G-Q217E, S063N-Q217E, S063R, S063R-Q217E, and S063R-Q217L, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and equal to or less than 1.0 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

The following BPN′-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in the BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16° C.: BPN′-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of D099N, K141Y-E156Q, N062L-S063L-Q217L, N062L-S063N, N062L-S063N-Q217E, N062L-S063R, N062L-S063R-Q217L, N062S-Q217E, N062S-S063G-Q217E, N062S-S063G-Q217R, N062S-S063N-Q217R, S063G-S125A, D060G-Q217L,

D120N-K141Y-K213Q, K043T-D099N-D120N-K141Y, K043T-D099N-K141Y-K256Q, K043T-K237A, N062L-S063G-Q217R, N062L-S063G-S125A, N062L-S063L-Q217E, N062L-S063N-S125A-Q217L, N062S-Q217R, N062S-S063L-Q217E, N062S-S063R-Q217L, S063G-M222S, S063G-Q217R, D120N-E156Q-K256Q, K141Y-D197N, N062L-Q217R, N062L-S063G-Q217L-M222S, N062L-S063L-Q217R, N062L-S063N-Q217R, N062S-Q217G, N062S-S063G-Q217G, N062S-S063G-Q217L-M222L, N062S-S063G-S125A-Q217L, N062S-S063N-Q217E, Q217G, S033G-N062S-S063G, S063G-Q217G, S063G-Q217L-M222L, S063G-S125A-Q217R, S063L-Q217R, S063N-M222S, S063N-Q217R, S063N-S125A-Q217L, S063R-Q217R, S063R-S125A-Q217L, D099N-E156Q-K256Q, E156Q, K012T-D099N-K213Q, K012T-K256Q, K043T-D099N-K141Y-K213Q, K043T-E156Q, K141Y-K213Q, N062L-Q217G, N062L-Q217L-M222L, N062L-Q217L-M222S, N062L-S063G-M222S, N062L-S063G-Q217L-M222L, N062L-S063G-Q217R-M222S, N062L-S063N-Q217L-M222S, N062L-S063N-S125A, N062L-S063R-S125A, N062L-S125A, N062S-S063G-M222S, N062S-S063G-Q217G-M222S, N062S-S063G-S125A, N062S-S063N-Q217L-M222L, N062S-S063N-S125A-Q217L, N062S-S063R-Q217G, N062S-S063R-Q217L-M222S, Q217G-M222S, Q217L-M222S, Q217R, S033G-S063G-Q217R, S063G-Q217E-M222S, S063G-S125A-Q217G, S063L-Q217E, S063N-Q217G, S063N-Q217G-M222S, S063N-Q217L-M222S, S063R-Q217L-M222S, and S063R-S125A, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2. Such variants have proteolytic activity. The invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN′-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence. Also included are compositions, including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such protease variant as described in greater detail elsewhere herein.

Part II Methods for Making GG36 Cold Water Protease Variants

A variety of methods are known in the art that are suitable for generating modified polynucleotides of the invention that encode GG36 cold water protease variants, including, but not limited to, for example, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches. Non-limiting exemplary methods of making the Series I GG36 cold water protease variants are provided in the section above entitled “Vectors, Cells, and Methods for Making Protease Variant Polypeptides of the Invention.”

Example 19

For testing of enzyme activity in heat-inactivated detergents, working solutions of detergents are made from the heat inactivated stocks. Appropriate amounts of water hardness (e.g., 6 gpg or 12 gpg) and buffer are added to the detergent solutions to match the desired conditions. The solutions are mixed by vortexing or inverting the bottles. The following Table provides information regarding some of the commercially-available detergents and test conditions used herein. In some experiments, additional and/or other commercially available detergents find use in the following Examples.

TABLE 19-1 Laundry and Dish Washing Conditions T Region Form Dose Detergent* Buffer Gpg pH (° C.) Laundry (Heavy Duty Liquid and Granular) NA HDL 0.78 g/l   P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0 g/L Henkel 5 mM HEPES 12 8.2 40 PERSIL ™ WE HDG 8.0 g/L P&G ARIEL ® 2 mM Na₂CO₃ 12 10.5 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 NA HDG 1.0 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 Automatic Dish Washing WE ADW 3.0 g/L RB 2 mM Na₂CO₃ 21 10.0 40 CALGONIT ™ NA ADW 3.0 g/L P&G 2 mM Na₂CO₃ 9 10.0 40 CASCADE ® In some additional Examples, the following solutions find use:

TABLE 19-2 Working Detergent Solutions Temp Detergent Detergent (C.) g/L pH Buffer Gpg TIDE ® 2X Cold 16 0.98 8 5 mM HEPES 6 TIDE ® 2X Cold 32 0.98 8 5 mM HEPES 6 TIDE ® 2X Cold 16 0.98 7 5 mM MOPS 6 Table 19-3 provides granular laundry detergent compositions produced in accordance with the invention suitable for laundering fabrics.

TABLE 19-3 Granular Laundry Detergent Compositions and Their Components Detergent Compositions Component 1 2 3 4 5 6 Linear 15 12 20 10 12 13 alkylbenzenesulfonate with aliphatic carbon chain length C₁₁-C₁₂ Other surfactants 1.6 1.2 1.9 3.2 0.5 1.2 Phosphate builder(s) 2 3 4 Zeolite 1 1 4 1 Silicate 4 5 2 3 3 5 Sodium Carbonate 2 5 5 4 0 3 Polyacrylate (MW 4500) 1 0.6 1 1 1.5 1 Carboxymethyl cellulose 1 — 0.3 — 1.1 — (Finnfix BDA ex CPKelco) Celluclean ® (15.6 mg/g) 0.23 0.17 0.5 0.2 0.2 0.6 Cold Water Protease 0.23 0.17 0.05 0.2 0.03 0.1 variant* Stainzyme Plus ® 0.23 0.17 0.5 0.2 0.2 0.6 (14 mg/g) Mannaway 4.0T (4 mg/g) 0.1 0.1 0.1 Lipex 100T (18.6 mg/g) 0.2 0.1 0.3 Fluorescent Brightener(s) 0.16 0.06 0.16 0.18 0.16 0.16 Diethylenetriamine 0.6 0.6 0.25 0.6 0.6 pentaacetic acid or Ethylene diamine tetraacetic acid MgSO₄ 1 1 1 0.5 1 1 Bleach(es) and Bleach 6.88 6.12 2.09 1.17 4.66 activator(s) Ethoxylated thiophene 0.002 0.001 0.003 0.003 — — Hueing Dye⁵ Direct Violet 9 ex Ciba 0.0006 0.0004 0.0006 Specialty Chemicals Sulfate/Citric Acid/ Balance to 100% Sodium Bicarbonate/ Moisture/perfume In Table 19-3, all enzyme levels expressed as % enzyme raw material, except for cold water protease variant (of this invention) which is expressed as % of active protein added to the product. Table 19-4 provides granular laundry detergent compositions suitable for top-loading automatic washing machines (detergent compositions 7-9) and front loading washing machines (detergent compositions 10-11). The GG36 protease variant tested and/or BPN′ variant and/or cold water protease variant of the present invention is added separately to these formulations.

TABLE 19-4 Granular Laundry Detergent Compositions and Their Components Component Detergent Composition Surfactants 7 8 9 10 11 C₁₆₋₁₇ Branched alkyl sulfate 3.55 15.8 C₁₂₋₁₄ alkyl sulphate 1.5 Sodium linear alkylbenzenesulfonate 9.6 10.6 7.5 9 with aliphatic chain length C₁₁-C₁₂ Sodium C_(14/15) alcohol ethoxy-3- 1.15 2.88 sulfate Sodium C_(14/15) alkyl sulphate 2.37 C_(14/15) alcohol ethoxylate with average 7 1.17 1 moles of ethoxylation mono-C₈₋₁₀ alkyl mono-hydroxyethyl di- 0.45 methyl quaternary ammonium chloride Di methyl hydroxyl ethyl lauryl 0.18 ammonium chloride Zeolite A 13.9 4.7 0.01 2.9 1.8 Sodium Silicate 1.6.ratio 4 0.2 4 4 Sodium Silicate 2.35.ratio 8 Citric Acid 2.5 1.4 Sodium tripolyphosphate 5 Sodium Carbonate 24.1 30 16.9 24.4 21 Nonanoyloxybenzenesuplhonate 5.78 2.81 0.96 Oxaziridinium-based bleach booster 0.03 0.017 Tetrasodium S,S,- 0.2 ethylenediaminedisuccinate Diethylenetriamine penta (methylene 0.61 0.33 phosphonic acid), heptasodium salt Hydroxyethane dimethylene phosphonic 0.29 0.45 acid Ethylene diamine tetraacetate 0.27 MgSO4 0.47 0.5994 0.782 Sodium Percarbonate 7 4.4 15.9 19.1 Tetra Acetyl Ethylene Diamine 3.3 4.6 Sodium Perborate Monohydrate 1.2 Carboxymethyl cellulose 0.1 0.17 1.69 0.23 (e.g., Finnfix BDA ex CPKelco) Sodium Acrylic acid/maleic acid co- 0.0236 3.8 2 2.5 polymer (70/30) Sodium polyacrylate (Sokalan PA30 CL) 4 0.84 Terephthalate polymer 0.23 Polyethylene glycol/vinyl acetate 0.89 0.89 0.91 random graft co polymer Photobleach- zinc phthalocyanine 0.005 0.001 0.002 tetrasulfonate C.I. Fluorescent Brightener 260 0.11 0.15 0.04 0.23 0.15 C.I. Fluorescent Brightener 351 0.1 (Tinopal ® CBS) Suds suppressor granule 0.25 0.07 0.04 Hydrophobically modified carboxy 0.019 0.028 methyl cellulose (Finnifix ® SH-1) Bentonite 8.35 Miscellaneous (Dyes, perfumes, process Balance Balance Balance Balance Balance aids, moisture and sodium sulphate)

In Table 19-4, surfactant ingredients can be obtained from any suitable supplier, including but not limited to BASF (e.g., LUTENSOL®), Shell Chemicals, Stepan, Huntsman, and Clariant (e.g., PRAEPAGEN®). Zeolite can be obtained from sources such as Industrial Zeolite. Citric acid and sodium citrate can be obtained from sources such as Jungbunzlauer. Sodium percarbonate, sodium carbonate, sodium bicarbonate and sodium sesquicarbonate can be obtained from sources such as Solvay. Acrylate/maleate copolymers can be obtained from sources such as BASF. Carboxymethylcellulose and hydrophobically modified carboxymethyl cellulose can be obtained from sources such as CPKelco. C.I. Fluorescent Brightener 260 can be obtained from 3V Sigma (e.g., OPTIBLANC®, OPTIBLANC® 2M/G, OPTIBLANC® 2MG/LT Extra, or OPTIBLANC® Ecobright. Tetrasodium S,S-ethylenediamine disuccinate can be obtained from sources such as Innospec. Terephthalate co-polymer can be obtained from Clariant (e.g., REPELOTEX SF 2). In addition, 1-Hydroxyethane -1,1-diphosphonic acid can be obtained from Thermphos. Oxaziridinium-based bleach booster has the following structure, where R1=2-butyloctyl, and was produced according to US 2006/0089284A1.

The enzymes NATALASE®, TERMAMYL®, STAINZYME PLUS®, CELLUCLEAN® and MANNAWAY® can be obtained from Novozymes. Zinc phthalocyanine tetrasulfonate can be obtained from Ciba Specialty Chemicals (e.g., TINOLUX® BMC). Suds suppressor granule can be obtained from Dow Corning. In these detergent compositions, random graft copolymer is a polyvinyl acetate grafted polyethylene oxide copolymer having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is about 6000 and the weight ratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60 and no more than 1 grafting point per 50 ethylene oxide units.

Assays and Test Methods

This Example describes the various assays used and provides further details of Test Methods 4-6.

TCA Assay for Protein Content Determination in 96-well Microtiter Plates

For GG36 and GG36 variants, this assay was started using filtered B. subtilis culture supernatants from microtiter plates grown 2-3 days at 37° C. with shaking at 250 rpm and humidified aeration. A fresh 96-well flat bottom microtiter plate (MTP; Costar 9017 medium binding clear polystyrene plate) was used for the assay. First, 100 μL/well of 0.25 N HCl was placed in each well. Then, 20-25 μL of filtered culture supernatant were added and the solution was mixed on a table top mixer (e.g., Lab line Instruments, Titer plate shaker, model 4825) for 5-10 seconds. The light scattering/absorbance at 405 nm was then determined, in order to provide the “blank” reading. For the “test” reading, 100 μL/well of 30% (w/v) trichloroacetic acid (TCA) was added to each well containing the mixture of HCl and culture supernatant, and the plate was incubated for 10 minutes at room temperature. After briefly mixing the solution on a table top mixer for no more than 2-3 sec, the light scattering/absorbance at 405 nm was determined. The turbidity/light scatter increase in the samples correlates to the total amount of precipitable protein in the culture supernatant. The calculations were performed by subtracting the “blank” reading (obtained after addition of HCl only, no TCA) from the “test” reading (obtained after addition of TCA, as described above) to provide a relative measure of the protein content in the samples. If desired, a standard curve can be created by calibrating the TCA readings with AAPF assays of clones with known conversion factors. However, the TCA results are linear with respect to protein concentration from 50 to 500 parts per million (ppm) of protein (where 1 ppm corresponds to 1 mg/L) and can thus be plotted directly against enzyme performance for the purpose of choosing variants with desired performance.

AAPF Protease Assay in 96-well Microtiter Plates

In order to determine the protease activity of the serine protease variants, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mM Tris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer); 100 mM Tris buffer, pH 8.6, containing 1 mM CaCl₂ and 0.005% TWEEN®-80 (Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1 ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer and mixed well for at least 10 seconds. The assay was performed by adding 10 μl of diluted protease solution to each well of a 96-well MTP, immediately followed by the addition of 190 μl of 1 mg/ml suc-AAPF-pNA working solution. The solutions were mixed for 5 sec, and the absorbance change in kinetic mode (25 readings in 5 minutes) was read at 405 nm in an MTP reader, at 25° C. The protease activity was expressed as AU (activity=ΔOD·min⁻¹ ml⁻¹).

Eglin C Inhibition Assay

As described herein, serine protease concentration and specific activity was determined by titration with an inhibitor called eglin c. Eglin c from the leech Hirudo medicinalis is a tight-binding protein inhibitor of subtilisins and ASP protease (Heinz et al., Biochemistry, 31: 8755-66 [1992]), and can therefore be used to measure protease enzyme concentration, which in turn permits specific activity to be calculated. The gene for eglin c was synthesized and expressed in E. coli by standard methods. Its properties and inhibitory potency were the same as eglin c purchased from Sigma.

(i) Concentration Determination of an Eglin C Stock Solution

A sample of Bacillus lentus subtilisin of known specific activity was diluted in 100 mM Tris buffer, pH 8.6, containing 1 mM CaCl₂ and 0.005% TWEEN®-80 (Tris/Ca buffer), to a concentration appropriate for AAPF protease assay described above. Several dilutions of the eglin c stock solution were also made in the Tris/Ca buffer. An aliquot of each diluted eglin c solution was mixed with an equal volume of the diluted Bacillus lentus subtilisin solution. An aliquot of the Tris/Ca buffer only, without eglin c, was also mixed with an equal volume of the diluted Bacillus lentus subtilisin solution, in order to measure uninhibited subtilisin activity in the absence of eglin c. The mixed solutions were incubated at room temperature for 15-30 minutes and the protease activity of each sample was then measured by AAPF assay described above. Using the known specific activity of Bacillus lentus subtilisin, the concentration of active protease in each sample was determined. The concentration of eglin c in each sample was then calculated based on the decrease of the observed protease activity as compared to the uninhibited subtilisin sample that was mixed with Tris/Ca buffer only (without eglin c). Thus, using the known dilutions and volumes of the eglin c solutions, the concentration of eglin c in the stock solution was determined.

(ii) Concentration and Specific Activity Determination of Subtilisin Variants

Samples of subtilisin variants were diluted in 100 mM Tris buffer, pH 8.6, containing 1 mM CaCl₂ and 0.005% TWEEN®-80 (Tris/Ca buffer). Several dilutions of the eglin c stock solution of known concentration were also made in the Tris/Ca buffer. An aliquot of each diluted eglin c solution was mixed with an equal volume of a subtilisin variant solution. The mixed solutions were incubated at room temperature for 15-30 minutes and the protease activity of each sample was then measured by AAPF assay. Using the observed decrease of the protease activity upon addition of each eglin c sample and the known concentration of the eglin c, the concentration of the eglin c necessary for the complete inhibition of each subtilisin enzyme variant was calculated. This concentration is equivalent to the enzyme concentration in the sample. An aliquot of the Tris/Ca buffer only, without eglin c, was also mixed with each subtilisin variant sample and the protease activity in the absence of eglin c was measured by AAPF assay. The specific activity of the subtilisin variants was then calculated using the enzyme concentrations as determined above.

BMI Microswatch Assay of Test Methods 4-6

Blood milk and ink (BMI) stained microswatches (EMPA116) of 5 5 millimeter circular diameter were obtained from CFT. In one method, the EMPA116 BMI fabric is pre-rinsed in water prior to cutting them into a 96we11 microtiter plate (Corning 3641), one microswatch per well. In the second method the EMPA 116 cloth is cut directly into a 96 well microtiter plate (Corning 3641) where the swatches are then rinsed with two water washes. The rinses are carried out by adding 200 μl of Milli Q water to each well/swatch and mixing them on a table top mixer (Lab line instruments, Titer plate shaker, model 4825) for 15 minutes at a setting of 7. The wash liquor is removed and 200 μl of water is added again to the swatch for another 15 minute rinse. The wash water is removed and the swatches are then air dried in the microtiter plate.

Detergent compositions 7-11 (Table 19-4) were diluted in Milli-Q (deionized) water to final working concentrations described in Table 19-1. These detergents were buffered with 2 mM sodium carbonate, pH 10.3. Additionally, a water hardness composition (3:1 Ca:Mg.—CaCl₂:MgCl₂.6H₂O) was added to each detergent solution to the final concentration described in Table 19-5. The detergent solutions were mixed at room temperature for 0.5 to 2 hours, centrifuged in 50 mL polypropylene conical tubes at 3000×g for 5-10 minutes and were kept at room temperature for the 32° C. assays or pre-equilibrated in an ice-water bath for the 16° C. assays. Then, 190 μl of the desired detergent solution was added to each well of the MTP containing BMI microswatches. To this mixture, 5-15 μl of the diluted enzyme master dilution solution were added, making the approximate concentration of enzyme in the reaction 0.25-2 μg/ml. The enzyme master dilution solution was prepared from the filtered culture supernatants (see TCA assay described above) at ˜2.5-20 μg/mL. The MTP was sealed with tape and placed in the iEMS incubator/shaker (Thermo/Labsystems) pre-set at 16° C. in a refrigerator for 30 minutes or at 32° C. on the benchtop for 30 minutes, with agitation at 1400 rpm. Following incubation under the appropriate conditions, 120-125 μl of the solution from each well was transferred into a fresh MTP (Corning 9017). The new MTP containing 125 μl of solution/well was read at 600 nm (with 5 sec mixing mode in the plate reader) using the MTP SpectraMax reader. Blank controls containing a microswatch and detergent without any enzyme were also included. The absorbance value obtained was corrected for the blank value (substrate without enzyme), providing a measure of hydrolytic activity. For each sample (variant) the performance index was calculated. The performance index compares the performance of the variant (measured value) and the standard enzyme (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of a performance dose response curve of the standard protease. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant as compared to the standard (e.g., wild-type), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard. Thus, the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.

TABLE 19-5 Final Detergent, Water Hardness, and Buffer Concentrations Used for BMI Microswatch Assays Final Sodium Final Detergent Final Water Carbonate Buffer Detergent Concentration Hardness* Concentration Composition (g/L) (gpg) (mM) 7 0.808 6 2 8 1 3 2 9 2.3 12 2 10 5.9 12 2 11 8.3 12 2

LAS/EDTA Stability Assay

The stability of protease variants in the presence of a representative anionic surfactant (LAS=linear alkylbene sulfonate, sodium dodecylbenzenesulfonate-DOBS) and di-sodium EDTA is measured after incubation under defined conditions and the residual activity is determined using the AAPF assay described above. The reagents used were dodecyllbenzene sulfonate, sodium salt (DOBS; Sigma No. D-2525), TWEEN®-80 (Sigma No. P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No. H-7523), unstressed buffer: 50 mM HEPES (11.9 g/l)+0.005% TWEEN®-80, pH 8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0, reference protease and protease variant culture supernatants, containing 200-400 μg/ml protein. The equipment used is V- or U-bottom MTP as dilution plates (Greiner 651101 and 650161 respectively), F-bottom MTP (Corning 9017) for unstress and LAS/EDTA buffer as well as for suc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (Molecular Devices), and iEMS Incubator/Shaker (Thermo/Labsystems).

The iEMS incubator/shaker (Thermo/Labsystems) is set at 29° C. Culture supernatants were diluted into plates containing unstress buffer to a concentration of ˜25 ppm (master dilution plate). For the assay, 20 μl of sample from the master dilution plate is added to plates containing 180 μl unstress buffer to give a final incubation concentration of 2.5 ppm. The contents were mixed and kept at room temperature and the AAPF assay is performed on this plate. In addition, 20 μl of sample from the master dilution plate is also added to plates containing 180 μl stress buffer (50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0). The solutions were mixed and immediately placed in 29° C. iEMS shaker for 30 min at 400 rpm. Following 30 minutes of incubation, the AAPF assay is performed on the stress plate. The stability of the samples is determined by calculating the ratio of the residual and initial AAPF activity as follows: Residual Activity (%)=[mOD·min−1 stressed]*100/[mOD·min−1 unstressed].

The final detergent, water hardness and buffer concentrations are determined based on the assay system to be used (e.g., North American, Japanese, Western European, or Central European conditions). In some aspects, the stain removal performance of the protease variants is determined in commercially available detergents. Heat inactivation of commercial detergent formulas serves to destroy the enzymatic activity of any protein components while retaining the properties of non-enzymatic components. Thus, this method is suitable for preparing commercially purchased detergents for use in testing the enzyme variants of the present invention.

Baked Egg Microtiter Assay

For this assay, 96-well baked egg yolk substrate plates are prepared from chicken egg yolks. Chicken egg yolks are separated from the whites, released from the membrane sac, and diluted 20% (vol/weight) with Milli-Q water. The diluted yolk is stirred for 15 min at room temperature using a magnetic stirrer. Five μL are carefully pipetted into the center of each well of a 96-well V-bottom plate (Costar #3894) using an 8-channel pipette. The plates are baked at 90° C. for 1 hour and cooled at room temperature. The baked egg yolk substrate plates are stored at room temperature and used within one week of preparation. Automatic dish detergents are prepared as described herein and pre-heated to 50° C. A 190 μL aliquot of detergent is added to each well of the 96-well plate using an 8-channel pipette. Ten μL of diluted enzyme is added to each well using a 96-channel pipetting device. The plate is carefully sealed with an adhesive foil sealer and incubated at 50° C. with shaking for 30 min. 120 μL of the reaction mixture is transferred to a new 96-well flat-bottom plate, and the absorbance/light scattering is determined at 405 nm. The absorbance/light scattering at 405 nm is proportional to egg yolk removal.

Performance Index

The performance index compares the performance of the variant (measured value) and the standard enzyme (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of a performance dose response curve of the standard protease. Various terms set forth below are used to describe the variant: non-deleterious variants have a PI>0.05; deleterious variants have a PI=0.05; combinable variants are those for which the variant has performance index values greater than or equal to 0.2 for at least one property, and >0.05 for all properties. Combinable variants are those that can be combined to deliver proteins with appropriate performance indices for one or more desired properties. These data find use in engineering any subtilisin/subtilase. Even if the subtilase to be engineered has an amino acid different from that of subtilisin GG36 at particular positions, these data find use in finding substitutions that will alter the desired properties by identifying the best choices for substitutions, including substitutions to the GG36 wild type amino acid.

Example 20 Generation of GG36 Single Mutants Using Site Evaluation Libraries (SELs)

The construction of GG36 SELs described in this example was performed by GENEART using their proprietary methods and technology platform for gene optimization, gene synthesis, library generation and analysis (WO 2004/059556A3, European Patent Nos. 0 200 362 and 0 201 184; and U.S. Pat. Nos. 4,683,195, 4,683,202 and 6,472,184). The GG36 SELs were produced at positions pre-selected by the inventors using the pHPLT-GG36 B. subtilis expression plasmid (see FIG. 6). This B. subtilis expression plasmid contains the GG36 expression cassette shown below, the B. licheniformis LAT promoter (Plat), and additional elements from pUB110 (McKenzie et al., Plasmid, 15:93-103, 1986) including a replicase gene (reppUB), a neomycin/kanamycin resistance gene (neo) and a bleomycin resistance marker (bleo) (FIG. 4 in U.S. Pat. No. 6,566,112). The pHPLT-GG36 plasmid map is provided at FIG. 6. The GG36 expression cassette sequence is provided below.

The DNA sequence of GG36 (the signal sequence is shown in lower case letters, propeptide in lower case, underlined text, and GG36 mature sequence in uppercase letters) is provided below:

(SEQ ID NO: 756) Gtgagaagcaaaaaattgtggatcgtcgcgtcgaccgcactactcatttc tgttgctttcagttcatcgatcgcatcggctgctgaagaagcaaaagaaa aatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaa caagtagaggcaaatgacgaggtcgccattctctctgaggaagaggaagt cgaaattgaattgcttcatgaatttgaaacgattcctgttttatccgttg agttaagcccagaagatgtggacgcgcttgagctcgatccagcgatttct tatattgaagaggatgcagaagtaacgacaatgGCGCAATCAGTGCCATG GGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAG GTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCA GACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCAC TCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTT TAAACAATTCGATTGGCGTTCTTGGCGTAGCGCCGAGCGCGGAACTATAC GCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGC CCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGA GTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGC GCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCAGGTGC AGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAG CTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGG CTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTC AACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAG GTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAA ATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTT GTATGGAAGCGGACTTGTCAATGCAGAAGCTGCAACTCGTTAA

The protein sequence of GG36 (the signal sequence is shown in lower case letters, propeptide in lower case, underlined text, and GG36 mature protease sequence in uppercase letters) is provided below:

(SEQ ID NO: 757) vrskklwivastallisvafsssiasaaeeakekyligfneqeavsefve qveandevailseeeeveiellhefetipvlsvelspedvdaleldpais yieedaevttmAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHP DLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELY AVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNS ATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAG LDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQ IRNHLKNTATSLGSTNLYGSGLVNAEAATR.

The method of mutagenesis was based on the codon-specific mutation approach in which all possible amino acid substitutions are simultaneously created at a specific codon of interest using forward and reverse mutagenesis primers that contain a degenerate codon, NNS ((A,C,T or G), (A,C,T or G), (C or G)) at the site of interest. To construct each of the GG36 SELs, three PCR reactions were performed: two mutagenesis reactions (primary PCR1 and PCR2) to introduce the mutated codon of interest in the mature GG36 DNA sequence using the NNS forward and reverse mutagenesis primers (25-45 nucleotides long), and a third reaction to fuse the two mutagenesis PCR products together to construct the pHPLT-GG36 expression vector having the desired mutated codons in the mature GG36 sequence.

The primer sequences used in this Example are provided below:

TABLE 20-1 Primers Sequence Primer Name CGCGCTTGAGCTCGATCCAGCGATTTC SacI-Fw (SEQ ID NO: 758) GTCTCCAAGCTTTAACGAGTTGCAG HindIII-Rv (SEQ ID NO: 759) GCAATTCAGATCTTCCTTCAGGTTATGACC pHPLT-BglII-Fw (SEQ ID NO: 760) GCATCGAAGATCTGATTGCTTAACTGCTTC pHPLT-BglII-Rv (SEQ ID NO: 761)

The Phusion High-Fidelity DNA Polymerase (Finnzymes catalog no. F-530L) was used for all PCRs, and the reactions were executed according to manufacturer's protocols that were supplied with the polymerase. In particular, for primary PCR 1, 1 μL (10 μM) of each of the pHPLT-BglII-Fw primer and a NNS reverse mutagenesis primer were used, and for primary PCR 2, 1 μL (10 μM) of the pHPLT-BglII-Rv primer and a NNS forward mutagenesis primer were used. Each reaction also included 1 μL of the pHPLT-GG36 plasmid template DNA (0.1-1 ng/μL).An MJ Research PTC-200 Peltier thermal cycler was used for the PCRs. The reactions yielded two fragments of approximately 2 to 3 kb having approximately 30 nucleotide overlap surrounding the GG36 codon of interest. The fragments obtained were fused in a third PCR similar to the ones described above using 1 μL of primary PCR 1 reaction mix, 1 μL of primary PCR 2 reaction mix and 1 μL (10 μM) of each of the forward and reverse SacI-Fw and HindIII-Rv primers. The amplified linear 859 bp fragment encoding the GG36 variant gene was purified (using QIAGEN® Qiaquick PCR purification kit) and digested with the Sad and HindIIII restriction enzymes to create cohesive ends on both sides of the fusion fragment. About 50 ng of plasmid pHPLT-GG36 was also purified after digestion with Sad and HindIIII, resulting in a 3.9 kb vector backbone fragment. The digested vector fragment was ligated with 50 ng of the digested 859 bp fragment encoding the variant enzyme using the T4 DNA ligase (Invitrogen) following the manufacturer's protocol for cloning of cohesive ends. Subsequently, the ligation mixture was used to transform B. subtilis cells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::[xylR,pxylA-comK]) as described (WO 2002/014490).

To express the variant proteins for further biochemical analyses, the B. subtilis strains carrying the GG36 variant plasmids were inoculated into microtiter plates containing 150 μl Luria broth medium supplemented with 10 μg/ml neomycin. Plates were grown overnight at 37° C. with 300 rpm shaking and 80% humidity using Enzyscreen lids for microtiter plates (Enzyscreen). Ten microliters from the overnight culture plate were used to inoculate a new microtiter plate containing 190 μl of MBD medium (a MOPS based defined medium) with 10 ug/ml neomycin. MBD medium was prepared essentially as known in the art (see Neidhardt et al., J. Bacteriol. 119:736-747 [1974]), except that NH₄Cl, FeSO₄, and CaCl₂ were omitted from the base medium, 3 mM K₂HPO₄ was used, and the base medium was supplemented with 60 mM urea, and 100 ml of a solution made of 210 g/L glucose, and 350 g/L maltodextrin. The micronutrients were made up as a 100× stock solution containing in one liter, 400 mg FeSO₄.7H₂O, 100 mg MnSO₄.H₂O, 100 mg ZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100 mg NaMo₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂, and 10 ml of 0.5 M sodium citrate. The MBD medium containing microtiter plates were grown for 68 hours at 37° C., 300 rpm, and 80% humidity using Enzyscreen lids (Enzyscreen) for determining protein expression. The next day, cultures were filtered through a micro-filter plate (0.22 μm; Millipore) and the resulting filtrate was used for biochemical analysis. The TCA and BMI microswatch assays for the detergent compositions 7-11 were carried out as described in Example 19. Performance indices were also calculated as described under the BMI assay of Test Methods 4-6 as described in detail in Example 19, and they are shown in Table 20-2 relative to GG36. In the following Tables, the detergent compositions (“Det.”) correspond to those shown in Table 19-4, above. Also, as indicated, the amino acid position is listed according to BPN′ numbering.

TABLE 20-2 Single Variants of GG36 with Performance Indices of at Least 0.2 Relative to GG36 in Either TCA or BMI Microswatch Cleaning at 16° C. in Detergents 7-11. GG36 Amino Acid Position (BPN′ Numbering) WT Residue Mutant Residue 1 A R 2 Q A 2 Q R 2 Q S 2 Q M 2 Q W 3 S R 4 V R 4 V S 4 V C 8 I A 9 S W 9 S F 9 S A 10 R A 10 R M 10 R S 10 R H 12 Q F 12 Q R 14 P F 14 P K 14 P Q 15 A R 15 A F 16 A S 17 H R 17 H F 17 H M 18 N R 18 N K 20 G R 20 G K 20 G F 22 T R 22 T Q 22 T L 22 T V 22 T W 22 T Y 22 T A 23 G A 23 G S 23 G F 24 S R 24 S W 24 S H 24 S L 24 S Q 24 S F 25 G R 25 G F 25 G V 26 V F 27 K R 27 K L 27 K V 27 K F 28 V A 28 V E 28 V N 29 A T 30 V E 31 L F 33 T S 33 T G 33 T D 34 G P 35 I M 36 S T 36 S F 36 S R 38 T R 38 T F 38 T L 40 P H 40 P W 40 P R 40 P N 40 P T 40 P L 42 L I 43 N R 43 N A 43 N S 43 N W 43 N F 43 N I 43 N D 43 N M 45 R T 46 G R 48 A R 50 F C 51 V W 51 V F 51 V H 52 P F 52 P N 52 P E 55 P Y 57 T R 59 Q A 59 Q F 59 Q R 60 D P 60 D A 60 D Q 62 N Q 62 N E 63 G S 63 G A 63 G M 63 G V 63 G T 63 G H 63 G Q 63 G I 63 G D 63 G E 63 G P 64 H F 64 H T 68 V A 68 V C 69 A N 69 A T 69 A W 69 A P 71 T G 72 I C 74 A C 75 L R 75 L A 75 L E 75 L F 78 S R 78 S I 78 S N 79 I Q 79 I W 81 V R 82 L R 82 L T 82 L M 82 L F 82 L V 85 A M 86 P W 86 P I 86 P L 89 E P 89 E W 89 E T 89 E I 89 E H 89 E V 89 E F 89 E L 89 E W 89 E G 91 Y F 91 Y N 92 A F 94 K N 99 S F 99 S T 99 S M 99 S G 99 S P 100 G I 100 G S 100 G N 100 G Q 101 S N 101 S G 101 S T 101 S A 101 S D 101 S F 101 S D 101 S E 101 S P 102 G A 102 G N 102 G T 102 G E 102 G H 103 S N 103 S G 103 S D 104 V L 104 V I 104 V E 104 V D 105 S T 105 S Q 105 S E 106 S V 106 S G 106 S T 106 S A 106 S E 106 S D 106 S F 107 I F 107 I M 108 A I 108 A G 109 Q M 111 L V 111 L I 112 E V 112 E L 112 E Q 114 A G 115 G R 115 G K 116 N L 116 N A 116 N K 117 N F 118 G I 118 G R 119 M C 120 H A 120 H F 120 H R 121 V E 121 V F 123 N G 123 N E 124 L S 128 S N 128 S M 128 S H 128 S Q 128 S I 128 S F 128 S L 128 S D 129 P E 132 S A 132 S E 138 A G 144 S R 147 V L 148 L I 158 A E 159 G C 159 G E 160 S D 166 S E 166 S D 167 Y W 175 M V 177 V C 181 D A 182 Q R 183 N D 183 N R 183 N I 183 N F 183 N M 185 N I 185 N E 185 N V 186 R H 186 R K 188 S R 188 S E 188 S D 192 Y W 192 Y H 194 A V 194 A F 194 A E 197 D F 198 I L 198 I F 203 V E 203 V C 208 T S 209 Y N 209 Y S 209 Y F 209 Y T 209 Y H 209 Y L 209 Y G 209 Y E 210 P V 210 P R 210 P L 211 G R 211 G Q 212 S I 212 S F 212 S M 213 T A 214 Y F 215 A F 215 A N 215 A H 215 A E 215 A D 216 S F 216 S A 217 L E 217 L N 217 L D 218 N P 218 N E 218 N D 224 T A 224 T G 227 V I 230 A E 231 A I 231 A C 233 L C 234 V F 235 K F 236 Q N 236 Q F 238 N R 238 N K 238 N L 239 P R 239 P S 239 P R 239 P H 239 P N 239 P K 239 P T 239 P F 239 P G 240 S R 241 W R 242 S R 242 S L 243 N R 243 N F 244 V R 246 I S 248 N I 248 N V 248 N R 249 H R 249 H T 250 L I 251 K R 251 K S 252 N R 252 N F 252 N H 252 N I 253 T R 253 T F 253 T I 254 A C 256 S N 258 G R 260 T V 260 T I 262 L H 262 L D 263 Y F 265 S F 267 L N 267 L M 267 L V 269 N R 269 N I 270 A C 271 E T 271 E V 271 E L 271 E H 271 E F 271 E P 271 E A 271 E M 271 E I 272 A F 272 A R 273 A I 273 A F 274 T G

TABLE 20-3 GG36 Single Variants with Performance Indices of at Least 1.5 Relative to GG36 BMI Microswatch Cleaning at 32° C. in Detergent 7. GG36 Variant N62E A158E G159E

TABLE 20-4 GG36 Single Variants with Performance Indices of at least 1.2 Relative to GG36 BMI Microswatch Cleaning at 32° C. in Detergent 10. GG36 Variant A1R S78R V244R N269R E271L

Example 21 Construction and Cleaning Performance of the NHJ1 and WCE1 Sets of GG36 Variants

The NHJ1 and WCE1 set of GG36 variants described herein were constructed at DNA 2.0, Inc., using the pHPLT-GG36 B. subtilis expression plasmid described above (FIG. 6). The variants were expressed in B. subtilis cells (genotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo) as described in Example 20, and were further characterized using the TCA assay for protein content determination, LAS/EDTA stability assay, and BMI microswatch cleaning assay as described in Example 19. These results are shown in Tables 21-1 and 21-2. In the following Tables, the detergent compositions (“Det.”) correspond to those shown in Table 19-4, above. Also, as indicated, the amino acid position is listed according to BPN′ numbering.

TABLE 21-1 NHJ1 Variants with Performance Indices of at least 0.25 Relative to GG36 in Any One of TCA, LAS/EDTA Stability, or BMI Microswatch Cleaning at 16° C. in Detergents 7, 8 or 9. GG36 Variant (BPN′ numbering) N062E-A158E S101A-H249R A016S-S101A S103G-A158E S188D-H249R E089P-S101A S128N-A158E V104L-H249R N062E-S103G A016S-A158E Y209E-H249R T022A-S103G V104L-A158E T022A-H249R A016S-S103G E089P-A158E S128N-H249R S101A-S103G L111V-A158E S103G-H249R E089P-S103G T022A-A158E E089P-H249R N062E-S128N S101A-A158E T022A-L111V A016S-S128N L148I-A158E S101A-L111V T022A-S128N P129E-A158E A016S-L111V S101A-S128N T022A-E089P V104L-L111V V104L-S128N A016S-E089P N062E-L111V E089P-S128N N062E-E089P S103G-L111V S103G-S128N N062E-E271F E089P-L111V L111V-S128N A158E-E271F A016S-L148I L111V-S188D R186H-E271F N062E-L148I N062E-S188D P129E-E271F T022A-L148I A016S-S188D L111V-E271F P129E-L148I L148I-S188D Y209E-E271F V104L-L148I T022A-S188D A016S-E271F S103G-L148I S128N-S188D S188D-E271F S128N-L148I S101A-S188D T022A-E271F S101A-L148I V104L-S188D G159E-E271F E089P-L148I E089P-S188D V104L-E271F L111V-L148I P129E-S188D S101A-E271F A016S-N062E G159E-S188D E089P-E271F T022A-N062E R186H-S188D S128N-E271F N062E-P129E S103G-S188D S103G-E271F T022A-P129E A158E-S188D L148I-E271F S128N-P129E A016S-T022A H249R-E271F A016S-P129E A016S-V104L N062E-G159E S101A-P129E T022A-V104L A016S-G159E V104L-P129E S101A-V104L S128N-G159E E089P-P129E N062E-V104L L148I-G159E S103G-P129E S103G-V104L L111V-G159E L111V-P129E E089P-V104L E089P-G159E N062E-R186H G159E-Y209E T022A-G159E S128N-R186H L111V-Y209E P129E-G159E S101A-R186H S101A-Y209E S103G-G159E T022A-R186H A016S-Y209E V104L-G159E A016S-R186H S128N-Y209E A158E-G159E A158E-R186H L148I-Y209E S101A-G159E E089P-R186H P129E-Y209E A158E-H249R P129E-R186H N062E-Y209E L111V-H249R G159E-R186H T022A-Y209E P129E-H249R S103G-R186H S103G-Y209E N062E-H249R V104L-R186H A158E-Y209E A016S-H249R L111V-R186H S188D-Y209E R186H-H249R L148I-R186H V104L-Y209E L148I-H249R N062E-S101A E089P-Y209E G159E-H249R T022A-S101A R186H-Y209E

TABLE 21-2 WCE1 Variants with Performance Indices of at least 0.2 Relative to GG36 in Any One of TCA, LAS/EDTA Stability, or BMI Microswatch Cleaning at 16° C. in Detergents 10 or 11. GG36 Variant (BPN′ Numbering) N018R-W241R G115R-S212F A001R-L082R G020R-W241R G020R-S212F S078R-L082R S024R-W241R S009A-S212F G020R-L082R S009A-W241R N043R-S212F T022R-L082R G020R-W241R S078R-S212F V004R-L082R V004R-W241R L082R-S212F N043R-L082R N043R-W241R S009A-S078R N043R-H249R S078R-W241R G020R-S078R G020R-H249R T022R-W241R S024R-S078R V004R-H249R G115R-W241R T022R-S078R N018R-H249R A001R-W241R N018R-S078R S009A-H249R S212F-W241R V004R-S078R S212F-H249R L082R-W241R A001R-S078R T022R-H249R N018R-V244R N043R-S078R S024R-H249R S024R-V244R T022R-S024R G115R-H249R S078R-V244R G020R-S024R A001R-H249R G020R-V244R N018R-S024R L082R-H249R S212F-V244R A001R-S024R S242R-H249R S009A-V244R V004R-S024R W241R-H249R L082R-V244R S009A-S024R V244R-H249R A001R-V244R V004R-S009A S078R-H249R N043R-V244R A001R-S009A N018R-G115R T022R-V244R S242R-N269R G020R-G115R V004R-V244R S024R-N269R T022R-G115R G115R-V244R G020R-N269R S078R-G115R W241R-V244R T022R-N269R S009A-G115R S242R-V244R H249R-N269R V004R-G115R A001R-V004R S212F-N269R A001R-G115R S009A-T022R N043R-N269R L082R-G115R N018R-T022R V244R-N269R N043R-G115R G020R-T022R A001R-N269R S024R-G115R V004R-T022R N018R-N269R S009A-G020R A001R-T022R S078R-N269R N018R-G020R S024R-S242R S009A-N269R V004R-G020R N018R-S242R G115R-N269R A001R-G020R V004R-S242R W241R-N269R S009A-E271L G020R-S242R V004R-N269R G020R-E271L S212F-S242R L082R-N269R S024R-E271L L082R-S242R N018R-N043R V244R-E271L S078R-S242R G020R-N043R W241R-E271L A001R-S242R V004R-N043R N043R-E271L S009A-S242R T022R-N043R T022R-E271L T022R-S242R S009A-N043R H249R-E271L G115R-S242R A001R-N043R S212F-E271L N043R-S242R S024R-N043R G115R-E271L W241R-S242R S009A-N018R S242R-E271L N018R-S212F V004R-N018R S078R-E271L T022R-S212F A001R-N018R V004R-E271L V004R-S212F S024R-L082R N269R-E271L S024R-S212F S009A-L082R A001R-E271L A001R-S212F N018R-L082R N018R-E271L L082R-E271L

Example 22 Construction and Cleaning Performance of NHJ4 Set of GG36 Variants

The NHJ4 set of GG36 variants described in Table 22-1 below were constructed using the pHPLT-GG36 B. subtilis expression plasmid (FIG. 6) using PCR fusion or the QUIKCHANGE® Multi Site-directed mutagenesis kit (“QCMS kit”; Stratagene) as described below.

a) Construction of NHJ4 Variants by QUIKCHANGE® Multi Site-Directed Mutagenesis

Variants created using the QUIKCHANGE® Multi Site-Directed Mutagenesis are shown in Table 22-1. The parent plasmid pHPLT-GG36 (template DNA) was methylated using two micrograms of DNA and Dam methylase (NEB), according to the manufacturer's instructions. Site-directed mutants were made by a QuikChange® Multi Site-Directed Mutagenesis Kit (“QCMS kit”; Stratagene) following the manufacturer's protocol. The following mutations were introduced in the parent plasmid

S101A-S103G-V104L, G159E, T22A, Y209E, E271F, S101A, S103G, L111V, S128N, N62E, and S188D, For efficient transformation of B. subtilis, DNA from the QCMS reaction mixtures was amplified by rolling circle amplification (RCA) using the Illustra Templiphi kit (GE Healthcare) and the reaction was performed according to the manufacturer's protocol. One microliter of ten-fold diluted amplified DNA was used to transform 50 μL of competent B. subtilis cells (genotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). The transformation mixture was shaken at 37° C. for 1 hour. Ten microliter aliquots of the transformation mixture were plated on skim milk (1.6%) Luria agar plates supplemented with 10 μg/ml of neomycin (Teknova). Subsequently, the colonies with halos were inoculated in 120 μl of LB media containing 10 μg/mL neomycin for plasmid DNA extraction (QIAprep Spin Miniprep kit, Qiagen). The extracted plasmids were sequenced to confirm the presence of the desired mutations.

b) Construction of NHJ4 Variants by Extension PCR

Ten combinatorial mutants of GG36 were created by extension PCR. The list of mutations introduced in the GG36 gene contained in the pHPLT plasmid were T22A, N62E, S103G, S103G-L111V, S101G-S103A-V104I, S101A-S103G-V104L, S101A, S128N, G159D, G159E, Y209E, and L111V. To create each mutant, PCR fragments containing the desired mutations were amplified using mutagenic primers as well as forward and reverse primers to amplify the entire GG36 variant. Each PCR amplification reaction contained 30 pmol of each mutagenic primer and 100 ng of the DNA template, pHPLT-GG36 plasmid Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 μL) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15 sec., annealing at 55° C. for 15 sec. and extension at 72° C. for 1 min. Following amplification, 2 to 4 PCR fragments for each variant were gel-purified, using a QIAGEN® gel-band purification kit and mixed (50 ng of each fragment). These mixtures served as DNA templates for the extension PCR to generate the full-length gene fragments. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min. The full-length DNA fragment was gel-purified using a QIAGEN® gel-band purification kit, digested with the BamHI and HindIII restriction enzymes and ligated with the pHPLT-GG36, which was digested with the same restriction enzymes. One microliter of the ligation mixtures was amplified using rolling circle amplification by Illustra Templiphi kit according to the manufacturer's instructions (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. Products of the rolling circle amplification were diluted 100-times and used to transform B. subtilis cells (genotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, the colonies with halos were inoculated in 120 μl of Luria broth medium containing 10 μg/mL neomycin for plasmid DNA extraction (QIAprep Spin Miniprep kit, Qiagen). The extracted plasmids were sequenced to confirm the presence of the desired mutations. Variants created by the extension PCR are shown in Table 22-1.

To express the NHJ4 set of variant proteins for further biochemical analyses, the B. subtilis strains carrying the variant plasmids were inoculated into microtiter plates containing 150 μl Luria broth medium supplemented with 10 μg/ml neomycin. The cultures were grown up for protein expression as described in Example 20, and they were filtered through a micro-filter plate (0.22 μm; Millipore) also as described in Example 20. The resulting filtrate was used for biochemical analysis. The eglin c inhibition assay for protein content determination and BMI microswatch assays tested in various detergents were carried out as described in Example 19. Performance indices are also calculated as described under the BMI assay description of Test Methods 4-6 further detailed in Example 19. Table 22-1 provides information regarding these multiple mutation variants and the results obtained for them. The PI values are relative to GG36. In the following Tables, the detergent compositions (“Det.”) correspond to those shown in Table 19-4, above. Also, as indicated, the amino acid position is listed according to BPN′ numbering.

TABLE 22-1 NHJ4 Multiple Mutation Variants with BMI Cleaning Performance Indices of at Least 0.2 Relative to GG36 in Detergents 7, 8 or 9 at 16° C. Variant Name Created by Mutations, (BPN′ Numbering) GG36 NHJ4-1 Extension PCR S101G S103A V104I NHJ4-10 Extension PCR T22A S101A Y209E NHJ4-11 Extension PCR S103G L111V G159E NHJ4-12 Extension PCR T22A S103G G159E NHJ4-13 QCMS T22A L111V G159E NHJ4-14 QCMS T22A S128N E271F Y209E NHJ4-15 QCMS T22A S103G L111V NHJ4-16 QCMS N62E L111V S128N NHJ4-17 QCMS T22A L111V S128N NHJ4-18 Extension PCR T22A N62E L111V NHJ4-19 QCMS S101A S103G V104L S188D NHJ4-2 Extension PCR S101G S103A V104I G159D NHJ4-20 Extension PCR S101A S103G V104L S128N NHJ4-24 QCMS T22A S101A G159E NHJ4-3 Extension PCR S101A S103G V104L NHJ4-4 Extension PCR S101A S103G V104L G159E NHJ4-5 Extension PCR T22A S101A S103G V104L NHJ4-6 QCMS S101A S103G V104L Y209E NHJ4-7 QCMS T22A Y209E E271F NHJ4-8 QCMS T22A S101A E271F NHJ4-9 QCMS S101A Y209E E271F

Example 23 Construction and Cleaning Performance of NHJ3 Set of GG36 Variants

The NHJ3 set of variants described herein are based on a variant of GG36 (referred to as GG36-9) containing the following mutations: S101G, S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, and N252K (BPN′ numbering). These variants were created using the QUIKCHANGE® Lightning Site-Directed Mutagenesis Kit (QCLDS kit; Stratagene), with the pRA68 plasmid (see FIG. 7) as the DNA template. Plasmid pRA68 was derived from the pBN3 vector (see Babe et al., Biotech. Appl. Biochem. 27:117-124 [1998]).

The DNA sequence of GG36-9 variant (the signal sequence is shown in lower case letters, propeptide in lower case, underlined text, and GG36-9 mature sequence in uppercase letters) is provided below:

(SEQ ID NO: 762) Gtgagaagcaaaaaattgtggatcgtcgcgtcgaccgcactactcatttc tgttgcttttagttcatcgatcgcatcggctgctgaagaagcaaaagaaa aatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaa caagtagaggcaaatgacgaggtcgccattctctctgaggaagaggaagt cgaaattgaattgcttcatgaatttgaaacgattcctgttttatccgttg agttaagcccagaagatgtggacgcgcttgaactcgatccagcgatttct tatattgaagaggatgcagaagtaacgacaatgGCGCAATCAGTGCCATG GGGAATTAGCCGTGTGCAAGCCCCGGCTGCCCATAACCGTGGATTGACAG GTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCA GACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCAC TCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTC TAAACAATTCGATTGGCGTACTTGGCGTAGCGCCGAGCGCGGAACTATAC GCTGTTAAAGTATTAGGGGCGAGCGGTGGGGGCGCCATCAGCTCGATTGC CCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGA GTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGC GCGACTTCTAGGGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCGGGTGC AGACTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAG CTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGG CTTGACATCGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTC AACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAG GTGCAGCAGTCCTTGTTAAACATAAGAACCCATCTTGGTCCAATGTACGA ATCCGCGATCATCTAAAGAAAACGGCAACGAGCTTAGGAAGCACGAACTT GTATGGAAGCGGACTTGTCAATGCCGAAGCTGCAACTCGTTAA

The protein sequence of the GG36-9 variant (the signal sequence is shown in lower case letters, propeptide in lower case, underlined text, and GG36-9 mature protease sequence in uppercase letters) is provided below:

(SEQ ID NO: 763) vrskklwivastallisvafsssiasaaeeakekvligfneqeavsefve qveandevailseeeeveiellhefetipvlsvelspedvdaleldpais yieedaevttmAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHP DLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELY AVKVLGASGGGAISSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNS ATSRGVLVVAASGNSGADSISYPARYANAMAVGATDQNNNRASFSQYGAG LDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKHKNPSWSNVR IRDHLKKTATSLGSTNLYGSGLVNAEAATR

To create the NHJ3 variants using the QCLSD kit, mutagenic primers were designed for each of the variants according to the manufacturer's protocol. The mutagenesis reaction for each variant consisted of 0.5 μl of 10× Buffer, 0.5 μL of pRA68 plasmid DNA (168 ng/μL), 0.5 μl forward mutagenic primer (25 μM), 0.5 μl reverse mutagenesis primer (25 μM), 1 μl dNTPs (supplied in the QCLSD kit), 1.5 ul Quik solution (supplied in the QCLMS kit), 1 μl Enzyme blend (supplied in the QCLSD kit), and 40 μl of distilled, deionized water to make up a 50 μL reaction volume as per the manufacturer's instructions. The cycling program was 1 cycle at 95° C. for 2 minutes, 18 cycles of 95° C. for 20 seconds, 60° C. for 10 seconds and 68° C. for 3 minutes, 22 seconds, and a final cycle of 68° C. for 5 minutes. Next, 1 μL of DpnI restriction enzyme supplied in the kit was used to digest the plasmid DNA in the reaction, and then 2 μL of the reaction was used to transform TOP 10 E. coli competent cells (Invitrogen). The E. coli transformants were selected on Luria broth medium plates containing 50 μg/mL(ppm) carbenicillin after overnight growth at 37° C. Plasmid DNA was extracted from 4-8 E. coli colonies grown in LA medium containing 50 μg/mL(ppm) carbenicillin using the QIAprep spin miniprep kit (Qiagen). The plasmids were sequenced to confirm the presence of the desired mutations. The variant plasmids were then transformed into B. subtilis cells as described in Example 20. The B. subtilis variant strains were grown up as described in Example 20 for further biochemical analysis, such as protein content determination using the eglin c inhibition assay (Example 19) and a BMI microswatch cleaning assay (Test Methods 4-6, Example 19). The results are provided below in Tables 23-1 and 23-2. The PIs are relative to GG36. In the following Tables, the detergent compositions (“Det.”) correspond to those shown in Table 19-4, above. Also, as indicated, the amino acid position is listed according to BPN′ numbering.

TABLE 23-1 NHJ3 Multiple Mutation Variants with BMI Cleaning Performance Indices of at Least 1.1 Relative to GG36 in Detergents 7, 8, or 9 at 16° C. Variant Variant Sequence Relative to GG36 (Using BPN′ Numbering) GG36 NHJ3-1 S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-2 S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-3 S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-4 S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K NHJ3-5 S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K NHJ3-6 S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K NHJ3-7 S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K NHJ3-8 S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K NHJ3-9 S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D

TABLE 23-2 NHJ3 Multiple Mutation Variants with BMI Cleaning Performance Indices of at Least 0.3 Relative to GG36 in Detergents 10 or 11 at 16° C. Variant Variant Sequence Relative to GG36 (Using BPN′ Numbering) GG36 NHJ3-1 S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-2 S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-3 S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K NHJ3-4 S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K NHJ3-5 S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K NHJ3-6 S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K NHJ3-7 S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K NHJ3-8 S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K NHJ3-9 S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D

Example 24 Construction and Cleaning Performance of NHJ5 Set of GG36 Variants

The NHJ5 set of variants described herein are based on a variant of GG36 (referred to as GG36-7) containing the following mutations: S101G, S103A, V104I, G159D, A232V, Q245R, N248D, and (BPN′ numbering). These variants were created using the QUIKCHANGE® Lightning Multi Site-Directed Mutagenesis Kit (“QCLMS kit”) with the pRA96 plasmid as the DNA template (see FIG. 8). The mutations incorporated included: H243R (H249R), E265F (E271F), D157E (D159E), A156E (A158E), A156E-D157G (A158E-D159E), T22A, N60E (N62E), N232R (N238R), D242R (D248R), T247R (T253R), S24R, N74D (N76D) {GG36 Numbering and BPN′ Numbering Shown in Parentheses.

The variants were generated using the methods described in Example 23. The B. subtilis variant strains were grown up as described in Example 20 for further biochemical analysis, such as protein content determination using the eglin c inhibition assay (Example 19) and the BMI microswatch cleaning assay (Example 19). The results are provided below in Table 24-1. The PI values are relative to GG36. In the following Tables, the detergent compositions (“Det.”) correspond to those shown in Table 19-4, above. Also, as indicated, the amino acid position is listed according to BPN′ numbering.

The DNA sequence of GG36-7 variant (the signal sequence is shown in lower case letters, propeptide in lower case, underlined text, and GG36-7 mature protease sequence in uppercase letters) is provided below:

(SEQ ID NO: 764) gtgagaagcaaaaaattgtggatcgtcgcgtcgaccgcactactcatttc tgttgcttttagttcatcgatcgcatcggctgctgaagaagcaaaagaaa aatatttaattggctttaatgagcaggaagctgtcagtgagtttgtagaa caagtagaggcaaatgacgaggtcgccattctctctgaggaagaggaagt cgaaattgaattgcttcatgaatttgaaacgattcctgttttatccgttg agttaagcccagaagatgtggacgcgcttgaactcgatccagcgatttct tatattgaagaggatgcagaagtaacgacaatgGCGCAATCAGTGCCATG GGGAATTAGCCGTGTGCAAGCCCCGGCTGCCCATAACCGTGGATTGACAG GTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCA GACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCAC TCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTC TAAACAATTCGATTGGCGTACTTGGCGTAGCGCCGAGCGCGGAACTATAC GCTGTTAAAGTATTAGGGGCGAGCGGTGGGGGCGCCATCAGCTCGATTGC CCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGA GTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGC GCGACTTCTAGGGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCGGGTGC AGACTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAG CTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGG CTTGACATCGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTC AACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAG GTGCAGCAGTCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACGA ATCCGCGATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTT GTATGGAAGCGGACTTGTCAATGCCGAAGCTGCAACTCGT

The protein sequence of GG36-7 variant (signal sequence is shown in lower case letters, propeptide in lower case, underlined text, and GG36-7 mature protease sequence in uppercase letters) is provided below:

(SEQ ID NO: 765) vrskklwivastallisvafsssiasaaeeakekvligfneqeavsefve qveandevailseeeeveiellhefetipvlsvelspedvdaleldpais yieedaevttmAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHP DLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELY AVKVLGASGGGAISSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNS ATSRGVLVVAASGNSGADSISYPARYANAMAVGATDQNNNRASFSQYGAG LDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAVLVKQKNPSWSNVR IRDHLKNTATSLGSTNLYGSGLVNAEAATR

TABLE 24-1 NHJ5 Multiple Mutation Variants with BMI Cleaning Performance Indices of at Least 0.6 Relative to GG36 in Detergents 7-11 at 16° C. Variant Variant Sequence Relative to GG36 (BPN′ Numbering) GG36 GG36-7 S101G-S103A-V104I-G159D-A232V-Q245R-N248D NHJ5-1 S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F NHJ5-2 S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N238R NHJ5-3 S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N248R NHJ5-4 S101G-S103A-V104I-G159D-A232V-Q245R-N248D-T253R NHJ5-5 S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R NHJ5-6 S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N76D NHJ5-7 S101G-S103A-V104I-G159E-A232V-Q245R-N248D-H249R NHJ5-8 S101G-S103A-V104I-G159E-A232V-Q245R-N248D-E271F NHJ5-9 S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R NHJ5-10 S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F NHJ5-11 T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- H249R NHJ5-12 T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- E271F NHJ5-13 N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- H249R NHJ5-14 N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D- E271F

Example 25 Construction of NHJ2 Combinatorial Library

This Example describes the construction of a GG36 combinatorial library involving one or more of the following mutations: A16S, T22A, S101A, S103G, V104L, L111V, S128N, and L148I (BPN′ numbering). The pHPLT-GG36 B. subtilis expression plasmid was provided to DNA 2.0 Inc. for the generation of NHJ2 combinatorial library. A ligation reaction of the constructed NHJ2 library was provided by DNA 2.0, Inc. for transformation in the B. subtilis strain (genotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). The variants generated containing one or several of the mutations described herein are tested for cold water cleaning applications using methods and detergent compositions described herein.

Example 26 Construction of Additional Libraries and GG36 Variants

Additional libraries and variants are constructed using the following set of mutations: A1R, A230E, E271L, G115R, G20R, H249R, K235F, K27V/F/L, L75E, L82R, N18R, N269R, N43D, N43R, N76D, R45T, S212F, S242R, S24R, S78R, S9A, T22R, V121E, V244R, V28E, V30E, V4R, W241R (BPN′ numbering). The variants generated containing one or more of these mutations are tested for cold water cleaning applications using methods and detergent compositions described herein.

Additional sets of GG36 variants are constructed and tested for cold water cleaning applications using methods and detergent compositions described herein include: G20R-N43R-H249R, G20R-T22R-N43R, G20R-N43R-S242R, G20R-N43R-E271L, G20R-N43R-V244R, G20R-S24R-N43R-S242R, S9A-T22R-S78R-S212F-W241R, S9A-G20R-N43R-S212F, S9A-N43R-S212F, G20R-N43R-S212F, G20R-T22R-N43R-S212F, S24R-S78R-S212F, S9A-N43R-S78R, S9A-N43R-S78R-S242R, S9A-G20R-N43R-S78R, G20R-S24R-N43R-S78R-S242R, T22R-S24R-S78R-S212F, S9A-G20R-N43R-S78R-S242R, G20R-N43R-S78R-H249R, G20R-N43R-S78R, S9A-S78R-S212F, S9A-T22R-N43R-S78R, S9A-G20R-S24R-N43R, S9A-T22R-S78R-S212F, V4R-S9A-T22R-S78R-S212F, G20R-S24R-N43R, A1R-S9A-N43R, G20R-S24R-N43R-G115R, S9A-S24R-N43R, G20R-T22R-S24R-N43R, A1R-S24R-N43R, S9A-G20R-S24R-N43R-S242R, S9A-G20R-T22R-S78R-S212F, S9A-S24R-N43R-V244R, S9A-S24R-N43R-S242R, V4R-S9A-T22R-S24R-S212F and T22R-S24R-N43R (BPN′ numbering).

Example 27 Construction and Cleaning Performance of the GG36 Library WCE2

The WCE2 combinatorial library was generated by DNA 2.0, Inc. using the pHPLT-GG36 B. subtilis expression plasmid. A ligation reaction of the constructed WCE2 library was provided by DNA 2.0, Inc. for transformation in the B. subtilis strain (genotype: ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). The set of mutations used to generate the WCE2 library are A230E, G20R, H249R, N18R, N43R/D, N76D, R45T, S242R, and S24R (BPN′ numbering). The variants generated containing one or more of these mutations are tested for cold water cleaning applications using methods and detergent compositions described herein.

Example 28 Construction and Cleaning Performance of the WCE3 Set of GG36 Variants

This Example describes the WCE3 set of mutants based on the GG36 variants, GG36-7 (Example 23) and GG36-9 (Example 22). These variants are: S101G-S103A-V104I-A232V-Q245R-N248D, S101G-S103A-V104I-G159D-A232V-Q245R, S101G-S103A-V104I-G159R-A232V-Q245R-N248D, S101G-S103A-V104I-G159D-A232V-Q245R-N248R, S101G-S103A-V104I-A232V-Q245R, S101G-S103A-V104I-A232V-Q245R-N248R, S101G-S103A-V104I-G159R-A232V-Q245R-N248R, and S101G, S103A, V104I, A232V, Q236H, Q245R, and N252K. They were created using the QuikChange® Lightning Multi Site-Directed Mutagenesis Kit (QCLMS kit; Stratagene) with the pRA96 plasmid as the DNA template described in Example 23. The variants generated will be tested for cold water cleaning applications using methods and detergent compositions described in this application.

Example 29 Construction of Additional Libraries and Variants of GG36

This Example describes the construction of GG36 variants and libraries using one or more of the following mutations: A16S, T22A, S24R, N62E, N76D, E89P, S101A/G, S103G/A, V104L/I, L111V, S128N, P129E, A232V, L148I, A158E, G159D/E, S166D, R186H, S188D, Y209E, Q236H, N238R, Q245R, N248D/R, H249R, N252K/R, T253R, E271F (BPN′ numbering) using a B. subtilis expression plasmid (e.g., pHPLT-GG36; FIG. 6). The variants generated containing one or more of these mutations are tested for cold water cleaning applications using methods and detergent compositions described herein.

Example 30 Construction of Additional Libraries and Variants of GG36

This Example describes the construction of GG36 variants and libraries in B. subtilis using one or more of the following mutations (BPN′ numbering): A1R, Q2S, Q2M, Q2A, Q2R, Q2W, S3R, V4R, V4S, V4C, I8A, S9A, S9F, S9W, R10S, R10A, R10H, R10M, Q12F, Q12R, P14K, P14F, P14Q, A15R, A15F, A16S, H17R, H17M, H17F, N18R, N18K, G20F, G20K, G20R, T22A, T22R, T22Y, T22V, T22Q, T22L, T22W, G23A, G23S, G23F, S24R, S24F, S24W, S24Q, S24H, S24L, G25V, G25F, G25R, V26F, K27L, K27F, K27R, K27V, V28A, V28N, V28E, A29T, V30E, L31F, T33S, T33G, T33D, G34P, I35M, S36T, S36F, S36R, T38L, T38F, T38R, P40N, P40L, P40T, P40W, P40H, P40R, L42I, N43A, N43F, N43I, N43S, N43R, N43M, N43W, N43D, R45T, G46R, A48R, F50C, V51W, V51F, V51H, P52F, P52E, P52N, P55Y, T57R, Q59A, Q59F, Q59R, D60P, D60Q, D60A, N62E, N62Q, G63V, G63M, G63T, G63I, G63A, G63S, G63H, G63Q, G63D, G63E, G63P, H64F, H64T, V68A, V68C, A69N, A69T, A69P, A69W, T71G, I72C, A74C, L75A, L75F, L75E, L75R, N76D, S78R, S78N, S78I, I79W, I79Q, V81R, L82F, L82T, L82V, L82R, L82M, A85M, P86W, P86L, P86I, E89P, E89T, E89G, E89H, E89L, E89V, E89W, E89F, E89I, Y91N, Y91F, A92F, K94N, S99F, S99T, S99P, S99G, S99M, G100S, G100N, G100Q, G100I, S101A, S101N, S101G, S101T, S101D, S101E, S101P, S101F, G102A, G102T, G102N, G102H, G102E, S103G, S103N, S103D, S103A, V104L, V104I, V104E, V104D, S105T, S105E, S105Q, S106G, S106T, S106E, S106D, S106A, S106V, S106F, I107M, I107F, A108I, A108G, Q109M, L111V, L111I, E112V, E112L, E112Q, A114G, G115K, G115R, N116K, N116A, N116L, N117F, G118R, G118I, M119C, H120A, H120F, H120R, V121F, V121E, N123G, N123E, L124S, S128D, S128F, S128L, S128N, S128H, S128M, S128I, S128Q, P129E, S132A, S132E, A138G, S144R, V147L, L148I, A158E, G159D, G159E, G159C, S160D, S166D, S166E, Y167W, M175V, V177C, D181A, Q182R, N183I, N183D, N183M, N183F, N183R, N185E, N185V, N185I, R186H, R186K, S188E, S188D, S188R, Y192H, Y192W, A194E, A194V, A194F, D197F, I198L, I198F, V203E, V203C, T208S, Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G, Y209L, P210R, P210V, P210L, G211Q, G211R, S212I, S212M, S212F, T213A, Y214F, A215N, A215D, A215E, A215H, A215F, S216F, S216A, L217E, L217N, L217D, N218D, N218P, N218E, T224A, T224G, V227I, A230E, A231I, A231C, A232V, L233C, V234F, K235F, Q236F, Q236N, Q236H, N238R, N238K, N238L, P239K, P239G, P239R, P239H, P239T, P239N, P239S, P239F, S240R, W241R, S242L, S242R, N243F, N243R, V244R, Q245R, I246S, N248D, N248V, N248I, N248R, H249R, H249T, L250I, K251R, K251S, N252I, N252F, N252R, N252K, N252H, T253I, T253R, T253F, A254C, S256N, G258R, T260V, T260I, L262D, L262H, Y263F, S265F, L267V, L267N, L267M, N269I, N269R, A270C, E271I, E271V, E271H, E271M, E271L, E271P, E271A, E271F, E271T, A272F, A272F, A272R, A273F, A273I, and T274G. The variants generated containing one or more of these mutations are tested for cold water cleaning applications using methods and detergent compositions described herein.

Example 31 Cleaning Performance Assay on BMI swatch at 60F (Test Method 2)

The stain removal performance of the protease variants was determined in commercially available detergents (unless otherwise stated). Heat inactivation of commercial detergent formulas serves to destroy the enzymatic activity of any protein components while retaining the properties of non-enzymatic components. Thus this method was suitable for preparing commercially purchased detergents for use in testing the enzyme variants of the present invention.

Microswatches:

Microswatches of ¼″ circular diameter were ordered and delivered by CFT (Vlaardingen, The Netherlands). Single microswatches or two microswatches were placed vertically in each well of a 96-well MTP to expose the whole surface area (i.e., not flat on the bottom of the well).

BMI Microswatch Assay

Microswatches containing blood milk and ink (BMI) of 0.25 inch circular diameter were obtained from CFT. Before cutting of the swatches, the fabric (EMPA 116) was washed with water. One microswatch was vertically placed in each well of a 96-well microtiter plate (hereinafter referred to as MTP) in order to expose the whole surface area (i.e., not flat on the bottom of the well). The desired detergent solution was prepared as described herein. After equilibrating the Thermomixer at 25° C., 190 μl of detergent solution was added to each well of the MTP, containing microswatches. To this mixture, 10 μl of the diluted enzyme solution was added so that the final enzyme concentration was 1 μg/ml (determined from TCA assay). The MTP was sealed with tape and placed in the incubator for 30 minutes, with agitation at 1400 rpm. Following incubation under the appropriate conditions, 100 μl of the solution from each well was transferred into a fresh MTP. The new MTP containing 100 μl of solution/well was read at 405 nm using a MTP SpectraMax reader. Blank controls, as well as a control containing two microswatches and detergent but no enzyme were also included.

“Pre-Washed” Swatch

This type of microswatch was pre-washed in deionised water for 20 minutes at ambient temperature. After the pre-washing step, the swatches were put on top of paper towels to dry. The air-dried swatches were then punched using a ¼″ circular die on an expulsion press. Finally two microswatches were put into each well of a 96-well MTP vertically to expose the whole surface area (i.e. not flat on the bottom of the well).

Detergents

For North American (NA) and Western European (WE) heavy duty liquid laundry (HDL) detergents, heat inactivation was performed by placing pre-weighed liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. All detergents were purchased from local supermarket stores. Both un-heated and heated detergents were assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity was tested by the AAPF assay.

For testing of enzyme activity in heat-inactivated detergents, working solutions of detergents were made from the heat inactivated stocks. Appropriate amounts of water hardness (for instance 6 gpg for NA HDL and 12 gpg for WE HDL) and buffer were added to the detergent solutions to match the desired conditions as specified in Table 31-1 below. The solutions were mixed by vortexing or inverting the bottles.

TABLE 31-1 Laundry and Dish Washing Conditions Region Form Dose Detergent* Buffer gpg pH T (° C.) Laundry (heavy duty liquid and granular) NA HDL 0.78 g/l   P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0 g/L Henkel Persil 5 mM HEPES 12 8.2 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 *Abbreviations: Procter & Gamble (P&G);

Enzymes and Equipment

Samples of reference serine proteases variants thereof were obtained from filtered culture broth of cultures grown in MTP plates. The equipment used was a Biomek FX Robot (Beckman Coulter), a SpectraMAX MTP Reader (type 340; Molecular Devices), an iEMS incubator/shaker (Thermo/Labsystems); F-bottom MTPs (Costar type 9017 used for reading reaction plates after incubation); and V-bottom MTPs (Greiner 651101 used for pre-dilution of supernatant). In this assay, the proteases hydrolyze the substrate and liberate pigment and insoluble particles from the substrate. Thus the rate of turbidity is a measure of enzyme activity.

The stain removal performance of reference serine proteases and variants therefrom on microswatches was determined on a MTP scale in commercially available heat-inactivated detergent. The reagents used were: 5 mM HEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer, 3:1 Ca:Mg for medium water hardness. (CaCl₂:MgCl₂.6H₂O); 15000 grains per gallon (gpg) stock diluted to 6 gpg, 2 BMI (blood/milk/ink) swatches per plate: EMPA-116 BMI cotton swatches processed by CFT: pre-rinsed and punched 2 swatches per well, and heat inactivated TIDE® 2× Cold off-the-shelf detergent in which lack of protease activity was confirmed.

TABLE 31-2 Working Detergent Solutions Temp Detergent Detergent (C.) g/L pH Buffer gpg Protease TIDE ® 2X 16 0.98 8 5 mM HEPES 6 BPN′ Cold TIDE ® 2X 32 0.98 8 5 mM HEPES 6 BPN′ Cold TIDE ® 2X 16 0.98 7 5 mM MOPS 6 BPN′ Cold

The incubator was set at the desired temperature (16° C. or 32° C.). 10 μL samples from the master dilution plate of ˜10 ppm enzyme was added to BMI 2-swatch plates with 190 μL working detergent solutions listed above. The volume was adjusted to give final concentration of 0.5 ppm for variants in the assay plates. The plates were immediately transferred to iEMS incubators and incubated for 30 minutes with 1400 rpm shaking at given temperature. Following incubation, 100 μL of supernatant was transferred into a new 96-well plate and the absorbance was measured in MTP Reader at 405 nm and/or 600 nm. Control wells, containing 1 or 2 microswatches and detergent without the addition of protease samples were also included in the test. The measurement at 405 nm provides a higher value and tracks pigment removal, while the measurement at 600 nm tracks turbidity and cleaning.

Calculation of the Stain Removal Activity for All Microswatch Assay Methods:

The absorbance value obtained was corrected for the blank value (substrate without enzyme), providing a measure of hydrolytic activity. For each sample (variant) the performance index was calculated. The performance index compares the performance of the variant (actual value) and the standard enzyme (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme.

Performance Index

The performance index compares the performance of the variant (actual value) and the standard or reference protease (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of the binding curve (i.e., Langmuir equation) of the standard protease. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant as compared to the standard (e.g., wild-type), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard. Thus, the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.

TCA Assay for Protein Content Determination in 96-well Microtiter Plates

For BPN′ (e.g., reference protease) and BPN′ variants, this assay was started using filtered culture supernatant from microtiter plates grown 3-4 days at 33° C. with shaking at 230 rpm and humidified aeration. A fresh 96-well flat bottom microtiter plate (MTP) was used for the assay. First, 100 μL/well of 0.25 N HCl was placed in each well. Then, 50 μL of filtered culture broth was added. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined, in order to provide the “blank” reading. For the test, 100 μL/well of 15% (w/v) trichloroacetic acid (TCA) was placed in the plates and incubated between 5 and 30 min at room temperature. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined.

The equipment used was a Biomek FX Robot (Beckman Coulter) and a SpectraMAX (type 340; Molecular Devices) MTP Reader; the MTP's were from Costar (type 9017). The equipment used was a Biomek FX Robot (Beckman Coulter) and a SpectraMAX type 340 (Molecular Devices) MTP Reader; and the MTPs were type 9017 (Costar).

The calculations were performed by subtracting the blank (no TCA) from the test reading with TCA to provide a relative measure of the protein content in the samples. If desired, a standard curve can be created by calibrating the TCA readings with AAPF assays of clones with known conversion factors. However, the TCA results are linear with respect to protein concentration from 50 micrograms to 500 micrograms protein per ml (ppm) and can thus be plotted directly against enzyme performance for the purpose of choosing good-performing variants. The turbidity/light scatter increase in the samples correlates to the total amount of precipitable protein in the culture supernatant.

AAPF Protease Assay in 96-well Microtiter Plates

In order to determine the protease activity of the proteases and variants thereof of the present invention, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (suc-AAPF-pNA) was measured. The reagent solutions used were: 100 mM Tris/HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer); 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl₂ and 0.005% TWEEN®-80 (Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a suc-AAPF-pNA working solution, 1 ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer and mixed well for at least 10 seconds. The assay was performed by adding 10 μl of diluted protease solution to each well, immediately followed by the addition of 190 μl 1 mg/ml suc-AAPF-pNA working solution. The solutions were mixed for 5 sec., and the absorbance change in kinetic mode (20 readings in 5 minutes) was read at 410 nm in an MTP reader, at 25° C. The protease activity was expressed as AU (activity=ΔOD·min⁻¹ ml⁻¹).

Stability Assay

The stability of protease variants was determined in the presence of 40% concentrated solution of detergent composition 3 shown in Table 1-3 diluted in water. The reagents used were the detergent composition 3 shown in Table 1-3 diluted to 50% in Milli-Q water, 10 mM MES 0.01% Tween 80 pH 5.8 master dilution buffer, AAPF reagents: see protocol AAPF assay. The equipment used was F-bottom MTP (Corning 9017) for dilution of diluted enzyme into detergent as well as for suc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), sealing tape: Nunc (236366), Circulating Reservoir (Beckman Fx).

The detergent composition 3 shown in Table 1-3 was initially diluted to 50% in water. This detergent was kept at room temperature and cycled through the circulating reservoir. The iEMS incubators/shakers (Thermo/Labsystems) were pre-set at 43°. Culture supernatants were diluted into plates containing master dilution buffer to a concentration of ˜20 ppm (master dilution plate). 40 μl of sample from the master dilution plate was added to plates containing 160 μl50% detergent composition 3 shown in Table 1-3 to give a final incubation concentration of 4 ppm. The contents were mixed and kept at room temperature and triplicate AAPF assays were performed immediately on these plates and recorded as unstressed reads. The AAPF assay was modified such that 20 μL of sample from the step above was added to 190 μL of suc-AAPF-pNA working solution. The plates were immediately covered with sealing tape and placed in 43° C. iEMS shakers for 30 min at 650 rpm. Following 30 minutes of incubation, triplicate AAPF assays were performed on these stress plates and recorded as stressed reads. The stability of the samples was determined by calculating the ratio of the residual and initial AAPF activity as follows: Residual Activity (%)=[mOD·min−1 stressed]*100/[mOD·min−1 unstressed].

LAS/EDTA Stability Assay

The stability of protease variants in the presence of a representative anionic surfactant (LAS=linear alkylbene sulfonate, sodium dodecylbenzenesulfonate-DOBS) and di-sodium EDTA was measured after incubation under defined conditions and the residual activity was determined using the AAPF assay. The reagents used were dodecyllbenzene sulfonate, sodium salt (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No. P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No. H-7523), unstress buffer: 50 mM HEPES (11.9 g/l)+0.005% TWEEN®-80, pH 8.0, Stress buffer: 50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0, reference protease and protease variant culture supernatants, containing 200-400 μg/ml protein. The equipment used was V- or U-bottom MTP as dilution plates (Greiner 651101 and 650161 respectively), F-bottom MTP (Corning 9017) for unstress and LAS/EDTA buffer as well as for suc-AAPF-pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), sealing tape: Nunc (236366).

The iEMS incubator/shaker (Thermo/Labsystems) was set at 29° C. Culture supernatants were diluted into plates containing unstress buffer to a concentration of ˜25 ppm (master dilution plate). 20 μl of sample from the master dilution plate was added to plates containing 180 unstress buffer to give a final incubation concentration of 2.5 ppm. The contents were mixed and kept at room temperature and a AAPF assay was performed on this plate. 20 μl of sample from the master dilution plate was also added to plates containing 180 μl stress buffer (50 mM HEPES (11.9 g/l), 0.1% (w/v) DOBS (1 g/l), 10 mM EDTA (3.36 g/l), pH 8.0). The solutions were mixed and immediately placed in 29° C. iEMS shaker for 30 min at 400 rpm. Following 30 minutes of incubation, a AAPF assay was performed on the stress plate. The stability of the samples was determined by calculating the ratio of the residual and initial AAPF activity as follows: Residual Activity (%)=[mOD·min−1 stressed]*100/[mOD·min−1 unstressed].

Performance Data of BPN′ Variants

The below tables exemplify the performance of the Series 1 BPN′ cold water protease variants described in this patent as assayed by Test Method 2.

Table 31-3: Performance of Single Mutation Proteases

-   Library Parent: BPN′ Wild type (Enzyme of SEQ ID NO:2) -   PI reference: calculated relative to BPN′ WT for the BMI pH8 16° C.     assay. -   Detergents: Commercial heat deactivated Tide 2× Cold

Description:

In this Example, results of experiments conducted to determine protein expression, stain removal activity, LAS stability, and AAPF activity (tests of properties of interest) of BPN′ and BPN′-variants are described. BPN′ single variants were constructed using PCR fusion as described in USPA 2009/46156, filed June 3, 2009, herein incorporated by reference. The results were obtained using the methods described in Test Method 2. As described throughout, functionality of the BPN′ variants was quantified as a performance index (PI), which is the ratio of performance of a variant to a parent protein. Table 32-1 provides Performance index values (PI) of subtilisin BPN′ variants. Performance indices less than or equal to 0.1 were fixed to 0.1 and indicated in bold italics in the table. Also, for the stability measure, if the Performance index of activity in the stability assays was less than or equal to 0.1, the associated stability performance index was fixed to 0.1.

TABLE 31-3 Performance Index Values for BPN′ Variants PI BMI PI BMI PI BMI LAS- specific BPN′ TCA pH 8 pH 7 pH 8 EDTA AAPF Position variant PI 16 C 16 C 32 C PI PI 2 Q002W 0.2 3.8 6.9 2.4

0.5 5 P005K 0.2 4.5 2.6 0.7 0.6 0.1 5 P005L 0.2 1.5 1.4 1.5

0.5 5 P005Y 0.2 3.3 3.3 2.2

0.4 7 G007T 0.2 3.0 2.4 3.0 0.1 0.2 8 V008G 0.3 1.4 0.5 1.4

0.4 8 V008K 0.2 3.1 3.0 3.1 0.2 0.5 8 V008P 0.2 2.7 3.0 2.9

0.6 11 I011G 0.2 2.2 2.2 1.6 0.4 0.3 11 I011H 0.2 36.5 114.2  1.9 0.8 0.4 11 I011S 0.3 1.3 1.7 1.3 0.3 1.0 12 K012L 0.2 3.1 4.6 2.0 0.6 0.3 13 A013M 0.2 1.4

0.8

16 L016W 0.2 3.4 6.2 3.2 0.1 0.4 23 G023S 0.2 10.8 4.1 2.6 1.1 0.1 26 V026H 0.5 1.5 0.9 1.2 1.1 0.9 26 V026W 0.3 2.3 1.2 2.1 1.1 1.0 26 V026Y 0.3 1.3 1.3 1.5 1.0 1.0 27 K027P 0.2 1.3 3.4 2.1 1.1 0.5 28 V028Q 0.2 2.8 2.0 1.4 1.6 0.3 28 V028S 0.2 5.7 5.2 3.4 1.1 0.4 28 V028T 0.6 1.3 1.2 1.1 1.0 1.2 29 A029C 0.8 1.3 1.2 1.1 0.9 0.8 29 A029D 0.2 3.0 2.7 2.3 0.9 0.4 29 A029S 0.5 1.6 1.1 1.2 1.1 1.0 29 A029T 0.2 2.0 1.6 1.6 1.1 0.5 29 A029V 0.4 1.9 1.5 1.6 1.0 0.8 30 V030D 0.2 2.3 2.3 2.0 0.9 0.2 30 V030E 0.2 30.2 33.3  4.3 0.8 0.2 30 V030G 0.2 7.6 3.7 1.4

30 V030T 0.3 1.4 1.3 1.4 0.9 0.4 31 I031E 0.2 2.6 2.0 2.4 1.1 1.1 31 I031G 0.2 5.4 3.7 2.4 1.1 0.4 31 I031H 0.3 2.0 1.6 1.8 1.3 1.2 31 I031K 0.3 1.8 1.3 1.4 1.2 1.0 31 I031N 0.2 2.3 2.0 2.0 1.3 0.7 31 I031Q 0.2 5.4 3.5 3.1 1.1 0.6 31 I031S 0.2 3.7 2.8 2.9 1.1 0.9 31 I031Y 0.2 4.1 3.0 3.1 0.9 0.9 33 S033F 0.5 1.4 0.6 1.0

33 S033H 0.5 1.7 0.8 1.2

36 D036L 0.3 1.9 1.3 1.4

0.7 37 S037P 0.5 1.5 0.9 1.1 0.4 0.7 41 D041A 0.2 2.5

0.8 0.1 0.2 41 D041C 0.3 1.7 0.3 1.1

0.5 41 D041M 0.2 10.4

0.6

41 D041N 0.3 2.1 1.3 1.4

0.5 41 D041S 0.2 3.1 1.5 1.2 0.5 0.3 42 L042S 0.2 32.8

0.4

42 L042Y 0.2 3.6 4.4 1.9

0.4 44 V044H 0.2 1.6 2.6 1.6 0.5 0.4 44 V044Q 0.2 1.5 2.9 1.5 0.6 0.3 44 V044T 0.3 1.3 1.9 1.3 0.7 1.3 46 G046F 0.4 1.4 1.0 1.0 1.0 0.9 46 G046L 0.3 1.6 1.4 1.4 0.8 0.8 46 G046M 0.4 1.3 0.8 1.1 0.9 1.0 46 G046V 0.2 4.9 3.3 2.1 0.8 0.7 47 G047T 0.2 4.5 4.8 2.3 0.5 0.1 47 G047W 0.2 2.6 2.4 2.2 0.5 0.2 49 S049I 0.2 2.2 2.5 2.1 0.5 0.1 49 S049V 0.2 1.6 1.4 2.1 0.4 0.3 50 M050D 0.2 6.4 0.9 0.9 1.0 0.2 50 M050I 0.3 1.6 0.9 1.1 0.9 0.6 50 M050R 0.3 1.4 0.7 1.1 1.0 0.8 51 V051A 0.4 1.4 1.3 1.2 0.9 0.4 51 V051G 0.2 3.4 4.3 1.4

51 V051S 0.3 1.8 2.0 1.4 0.9 0.4 52 P052C 0.3 1.4 1.6 0.9 1.0 0.4 52 P052I 0.5 1.3 1.5 1.1 0.8 0.6 52 P052L 0.5 1.3 1.2 1.3 0.8 0.6 52 P052M 0.4 1.4 1.3 1.1 0.7 0.5 52 P052V 0.3 1.8 1.6 1.0 1.0 0.5 52 P052W 0.4 1.5 1.3 1.4 1.0 0.8 52 P052Y 0.4 1.6 1.4 1.3 1.0 0.6 54 E054R 0.2 1.5 1.8 1.7 0.6 0.6 56 N056G 0.3 1.6 1.1 1.2 0.4 0.5 56 N056I 0.2 1.7 1.3 1.4 0.6 0.5 56 N056K 0.2 2.1 1.5 1.9 0.5 0.4 56 N056M 0.3 1.5 1.2 1.2 0.5 0.4 56 N056Q 0.3 1.7 1.3 1.2 0.8 0.4 56 N056R 0.2 4.9 4.0 2.2 0.5 0.3 56 N056V 0.3 1.4 1.3 1.0 0.5 0.5 56 N056Y 0.3 1.3 1.3 1.3 0.7 0.4 57 P057I 0.3 2.2 2.0 1.6 0.4 0.3 57 P057K 0.2 1.5 1.8 1.1 0.3 0.3 57 P057L 0.2 2.6 2.0 1.5 0.4 0.3 57 P057R 0.2 2.3 3.1 1.5 0.4 0.4 57 P057T 0.3 2.5 2.0 2.3 0.5 0.6 57 P057V 0.2 3.6 6.0 3.5 0.3 0.5 58 F058T 0.4 1.4 1.8 1.2 0.7 1.0 59 Q059P 0.3 1.9 2.0 1.5 0.8 0.4 59 Q059W 0.2 1.4 1.6 1.2 1.0 0.5 61 N061P 1.4 1.5 1.0 0.8 1.0 1.4 62 N062D 1.5 1.4 1.2 0.9 1.1 0.7 62 N062M 1.8 1.3 1.1 1.1 1.1 0.6 62 N062Q 1.5 1.3 1.2 1.1 1.2 0.6 63 S063C 1.6 1.3 1.0 0.7 1.3 0.8 63 S063Q 1.6 1.4 0.8 1.1 1.0 1.0 63 S063T 1.0 1.3 1.0 1.0 1.0 0.9 65 G065Q 0.2 1.7 2.1 2.3 1.2 0.7 68 V068A 1.0 1.8 1.1 1.3 1.0 0.1 68 V068G 0.5 1.8 1.3 1.4

68 V068M 1.2 1.3 0.9 1.2

68 V068S 1.3 1.7 1.3 1.1

69 A069C 0.4 1.7 0.9 1.2 0.7 0.5 69 A069D 0.3 2.0

69 A069F 0.2 13.7 1.3 0.5

69 A069H 0.2 39.0 2.2 0.7 0.7 0.1 69 A069M 0.2 7.2 0.7 0.6 0.5 0.1 69 A069N 0.2 2.5 0.1

69 A069P 0.3 1.8

69 A069Q 0.2 5.2 0.1

69 A069R 0.4 2.0

69 A069T 0.5 1.6 1.0 1.0 1.0 0.7 71 T071D 0.2 41.1 34.6  1.5 0.2 0.1 71 T071E 0.3 2.2 1.9 1.0 0.1 0.3 71 T071G 0.2 3.3 1.6 1.9

0.4 71 T071K 0.2 37.0 45.5  2.2 0.1 0.2 71 T071M 0.3 1.3 1.2 1.1

0.4 72 V072D 0.2 2.5 1.7 1.9 0.7 0.2 72 V072G 0.4 1.5 1.1 1.4 0.8 0.7 72 V072K 0.2 2.8 2.1 2.8 1.7 0.3 72 V072Q 0.3 1.6 1.5 1.7 0.6 0.6 72 V072S 0.4 1.4 1.4 1.3 0.8 0.9 72 V072T 0.6 1.4 1.0 1.1 0.9 1.0 73 A073E 0.5 1.6 1.2 1.1 0.2 0.8 73 A073I 0.2 22.5 15.7  6.1 0.1 0.3 73 A073K 0.2 3.5 2.5 2.6 0.1 0.3 73 A073M 0.2 1.7 1.4 1.5 0.3 0.4 73 A073Q 0.6 1.3 1.3 1.1 0.5 0.9 73 A073S 1.0 1.4 0.9 1.0 0.8 1.1 73 A073V 0.4 1.4 1.2 1.5 0.1 0.8 74 A074E 0.3 4.3

74 A074F 0.3 2.6

74 A074H 0.3 3.2

74 A074I 0.3 2.1

74 A074L 0.3 2.2

74 A074M 0.2 5.3

74 A074Q 0.3 2.9 0.1

74 A074R 0.3 1.9

74 A074V 0.3 3.6 0.1

74 A074W 0.3 2.5

74 A074Y 0.3 2.8 0.1

75 L075A 1.1 1.4 1.0 1.0

1.0 76 N076A 1.1 1.7 0.8 1.0

1.0 77 N077G 0.2 1.5 0.8 1.0

0.1 77 N077L 0.2 2.2 0.2 0.8 0.1 0.1 77 N077P 0.2 2.2

0.6 0.3 0.1 77 N077Q 0.2 2.1 0.8 1.2

0.4 77 N077R 0.2 2.5 0.1 0.9 0.1 0.2 77 N077S 0.3 1.7 1.3 1.2

0.8 77 N077T 0.2 4.2 1.3 1.5

0.3 78 S078W 1.0 1.3 0.7 1.0 0.3 1.1 80 G080H 0.2 11.3 32.2  3.1

0.3 81 V081D 0.2 1.4 0.8 0.6 0.1 0.1 81 V081F 0.9 1.3 0.9 1.1

0.8 81 V081H 0.7 1.5 1.0 1.0

0.7 81 V081N 0.4 1.3 0.9 1.2

0.7 81 V081Q 0.6 1.3 0.8 1.1

0.9 81 V081R 0.2 2.0 1.5 1.8

0.6 81 V081W 0.6 1.4 0.8 1.2

0.8 82 L082G 0.2 2.2 1.7 1.5 0.1 0.4 82 L082N 0.3 1.8 1.5 1.7

0.6 82 L082W 0.2 10.3 8.5 4.2 0.7 0.4 85 A085I 0.2 1.7 0.7 0.7

85 A085T 0.4 1.4 1.1 0.9 0.6 1.0 85 A085V 0.2 2.5 2.5 2.3 0.7 0.5 86 P086A 0.5 1.3 1.1 1.3 0.7 1.1 86 P086G 0.4 1.4 0.9 1.3 0.1 0.8 86 P086M 0.3 2.0 1.8 1.8 0.1 1.0 86 P086Q 0.3 1.5 1.6 2.0 0.1 0.9 86 P086R 0.2 3.5 3.8 2.7 0.7 0.7 86 P086T 0.3 1.7 1.8 1.9 0.2 1.1 86 P086W 0.4 1.4 1.5 1.3 0.4 1.4 86 P086Y 0.4 1.5 1.2 1.7 0.9 1.4 87 S087F 0.9 1.4 0.9 1.1 0.5 1.0 87 S087I 0.6 1.4 0.8 1.0 0.4 0.9 87 S087L 1.1 1.3 1.0 1.1 0.8 1.0 87 S087M 1.0 1.3 1.0 1.1 0.7 1.0 87 S087Q 0.8 1.3 0.9 1.0 0.8 0.9 87 S087V 0.9 1.3 1.0 1.2 0.9 1.2 87 S087W 0.9 1.4 1.0 1.3 0.5 1.1 88 A088D 0.3 1.4 1.6 1.3 0.7 0.7 88 A088E 0.2 11.4 17.5  1.7 0.6 0.2 88 A088K 0.2 2.4 3.0 1.7 0.8 0.7 88 A088P 0.2 2.2 3.4 2.0 0.9 0.6 88 A088Q 0.2 1.9 2.0 1.2 0.5 0.5 89 S089D 1.2 1.4 1.0 1.1 0.8 1.0 89 S089Q 0.2 108.1 37.2  0.2

89 S089V 0.7 1.3 1.3 1.2 0.7 1.3 90 L090D 0.2 2.4 2.1 2.2 0.6 0.5 90 L090E 0.2 1.6 1.4 1.5 1.0 0.5 90 L090F 0.3 1.6 0.8 1.4 0.3 0.3 90 L090H 0.2 2.0 2.2 2.3 1.0 0.9 90 L090P 0.3 1.6 1.7 1.6 0.9 0.9 90 L090S 0.2 1.6 1.9 1.6 0.7 0.9 90 L090T 0.2 3.9 3.3 2.5 0.8 1.0 91 Y091L 0.2 1.6 2.8 1.7 1.0 0.5 91 Y091Q 0.2 7.6 26.9  2.0 1.2 0.3 91 Y091T 0.2 3.7 8.1 2.7 0.9 0.6 92 A092C 0.3 2.6 2.0 1.7 0.6 0.3 92 A092I 0.3 1.7 1.5 1.6 0.7 0.3 92 A092M 0.2 2.5 2.6 2.9 0.6 0.2 92 A092N 0.2 6.4 7.3 5.5 0.7 0.2 92 A092P 0.3 1.6 2.3 2.1 0.9 0.5 92 A092V 0.3 1.4 1.3 1.7 0.6 0.5 93 V093D 0.2 2.4 3.4 2.0 1.1 0.7 93 V093F 0.2 1.3 2.2 1.5 1.0 0.6 93 V093L 0.6 1.4 1.1 1.0 0.8 1.0 93 V093T 0.3 1.5 1.7 1.4 1.1 1.0 94 K094C 0.2 1.3 1.4 1.1 1.0 0.2 94 K094R 0.4 1.4 1.5 1.3 0.8 0.4 94 K094S 0.2 4.2 3.8 2.5 1.0 0.2 95 V095I 0.4 2.4 1.2 1.3

96 L096F 0.5 2.0 0.8 1.4 0.9 0.6 96 L096H 0.2 1.9 0.2 0.4

96 L096I 0.8 1.6 0.9 1.0 1.2 0.4 96 L096M 0.9 1.5 1.1 1.2 0.9 0.5 96 L096N 0.2 6.1 0.9 0.7

96 L096Q 0.2 31.7 6.8 3.9

96 L096S 0.2 3.8 1.0 1.5

96 L096T 0.4 2.3 1.3 1.4

96 L096V 1.0 1.4 0.9 1.3 1.1 0.1 96 L096W 0.2 5.0 1.6 2.2 1.0 0.3 96 L096Y 0.3 2.0 0.7 1.2

97 G097A 0.9 1.6 1.0 1.3 1.0 0.8 97 G097C 1.0 1.7 1.1 1.1 1.0 0.5 97 G097D 1.6 1.4 0.8 1.2 1.0 0.8 97 G097E 1.1 1.4 0.8 1.1 1.1 0.7 97 G097F 0.3 1.9 1.0 1.4 0.9 0.3 97 G097L 0.8 1.4 1.1 1.2 1.1 0.7 97 G097M 0.8 1.3 1.0 1.2 1.1 0.9 97 G097P 0.4 2.4 1.6 1.3 1.1 0.7 97 G097Q 1.0 1.4 0.8 1.0 1.2 1.0 97 G097S 1.0 1.5 1.1 1.1 0.9 1.0 97 G097V 0.5 1.6 1.1 1.2 1.0 1.0 97 G097W 0.2 3.0 1.5 1.5 1.0 0.3 97 G097Y 0.4 1.4 0.9 1.0 0.9 0.3 99 D099C 0.7 1.5 0.8 1.1 1.1 0.6 99 D099E 1.0 1.4 0.8 1.0 1.0 0.8 99 D099I 0.4 1.6 0.6 1.1 1.0 0.6 99 D099M 0.6 1.4 0.7 1.2 0.9 0.7 99 D099P 0.4 2.0 0.9 1.2 1.0 0.4 99 D099V 0.4 1.7 0.8 1.1 0.9 0.6 99 D099Y 0.5 1.4 0.6 1.1 0.9 0.6 100 G100D 0.8 1.4 1.4 1.1 0.9 0.3 100 G100E 0.5 1.7 1.5 1.3 1.0 0.3 100 G100H 0.6 1.4 1.2 1.1 0.9 0.3 100 G100I 0.3 1.8 1.6 1.2

100 G100K 0.7 1.5 1.0 1.2 0.9 0.3 100 G100M 1.1 1.4 1.2 1.2 0.8 0.2 100 G100Q 0.7 1.6 1.2 1.4 1.0 0.4 100 G100T 0.3 2.2 1.6 1.5 1.0 0.2 100 G100V 0.3 2.0 1.6 1.4 1.0 0.1 100 G100Y 0.5 1.6 1.3 1.1 1.0 0.2 101 S101A 1.3 1.4 1.0 1.1 1.1 1.1 101 S101E 1.4 1.5 1.2 1.1 1.1 0.7 101 S101G 0.4 1.3 0.5 0.7 1.1 0.2 101 S101N 1.2 1.5 1.0 1.1 1.1 1.0 101 S101P 0.3 3.6 1.4 1.8 1.1 0.3 101 S101Q 0.6 1.6 0.8 1.1 1.0 1.2 101 S101T 0.9 1.3 0.7 1.2 1.0 1.1 101 S101V 0.9 1.4 0.7 1.2 0.9 1.2 102 G102A 1.1 1.5 1.3 1.2 1.0 0.3 102 G102S 0.8 1.6 1.1 1.0 1.1 0.1 103 Q103E 1.2 1.4 1.2 1.1 1.0 0.6 103 Q103G 0.4 1.5 1.1 1.3 1.1 0.4 103 Q103H 0.9 1.4 0.9 0.9 0.9 0.7 103 Q103K 0.2 2.6 1.4 1.3 1.0 0.5 103 Q103N 1.4 1.4 1.2 0.9 1.0 0.7 104 Y104L 0.2 1.5 1.5 1.2

104 Y104M 0.3 1.5 1.5 1.3 1.2 0.1 104 Y104N 0.5 1.4 1.8 1.0 1.1 0.1 104 Y104T 0.4 1.3 1.7 1.4 2.0 0.1 104 Y104V 0.3 1.7 1.2 1.4

105 S105D 0.4 1.3 1.0 1.2 1.1 0.9 105 S105I 0.2 1.6 1.1 0.7 1.0 0.3 105 S105R 0.2 3.0 3.4 1.0 1.2 0.2 105 S105V 0.2 2.8 3.1 2.8 1.0 0.8 106 W106A 0.4 1.4 1.2 1.1 0.9 0.4 106 W106C 0.4 1.4 1.1 1.0 0.9 0.2 106 W106E 0.4 1.7 1.3 1.2 1.1 0.4 106 W106F 0.8 1.3 0.8 1.3 1.0 0.4 106 W106G 0.2 1.4 1.3 1.2 0.9 0.1 106 W106I 0.3 1.5 1.1 1.3 1.1 0.4 106 W106L 0.3 1.6 1.1 1.4 1.1 0.3 106 W106M 0.4 1.4 1.1 1.1 0.9 0.3 106 W106S 0.4 1.4 0.9 1.2 2.0 0.2 106 W106T 0.4 1.4 1.2 1.3 0.9 0.3 106 W106V 0.3 1.5 1.2 1.2 0.9 0.5 107 I107R 0.2 2.3 2.3 1.9 1.2 0.1 107 I107S 0.5 1.4 0.9 1.2 1.0 0.1 107 I107T 0.7 1.4 2.3 0.9 1.0 0.1 108 I108S 0.2 2.4 8.6 1.3 0.9 0.1 108 I108T 0.3 1.8 1.7 1.5 0.9 0.8 110 G110S 0.3 2.2 1.4 1.7 0.9 0.5 110 G110T 0.2 4.6 4.0 2.8 0.9 0.5 111 I111A 0.2 3.8 2.4 1.9 0.9 0.3 111 I111C 0.4 1.9 1.5 1.5 0.9 0.9 111 I111F 0.5 1.7 1.1 1.2

111 I111L 1.0 1.3 1.0 1.1 1.0 0.7 111 I111M 0.8 1.4 1.0 1.0 1.1 0.7 111 I111T 0.3 3.1 1.6 1.7 1.0 0.9 111 I111V 0.7 1.6 1.0 1.3 1.0 0.8 112 E112I 0.2 7.2 2.7 2.7 1.1 0.6 112 E112L 0.2 19.7 11.6  3.8 1.1 0.6 112 E112T 0.3 2.9 2.1 2.0 1.0 0.9 113 W113H 0.2 1.5 1.7 1.5 1.1 0.6 114 A114I 0.2 3.4 2.6 2.5 0.9 0.4 114 A114V 0.3 1.6 2.2 1.6 0.9 1.0 115 I115A 0.4 1.6 1.0 1.1 0.9 0.8 115 I115E 0.3 2.3 1.7 1.6 0.9 0.7 115 I115F 0.3 2.0 1.2 1.4 0.9 0.6 115 I115H 0.2 4.9 2.5 2.4 0.9 0.5 115 I115M 0.9 1.5 1.0 1.1 1.0 0.9 115 I115N 0.2 8.3 8.4 2.6 1.0 0.4 115 I115P 0.2 1.8 0.1 0.2

115 I115Q 0.6 1.3 0.8 1.2 1.0 1.0 115 I115R 0.4 1.4 1.0 1.2 1.0 0.9 115 I115S 0.2 4.2 2.4 2.2 0.9 0.7 115 I115T 0.6 1.5 0.9 1.1 0.9 0.9 115 I115V 0.8 1.4 1.0 1.3 0.9 1.6 115 I115Y 0.2 4.8 4.1 2.5 0.9 0.8 117 N117K 0.2 1.9 1.9 1.8 1.0 0.9 117 N117V 0.2 5.4 4.1 2.1 1.0 0.5 117 N117W 0.3 2.1 1.8 1.6 1.0 1.1 118 N118I 0.2 4.9 5.9 2.0 1.1 0.6 118 N118L 0.2 1.5 1.9 1.3 1.0 1.1 118 N118V 0.2 1.4 1.9 1.5 1.0 1.0 119 M119F 0.3 1.6 1.9 1.6 1.3 0.8 119 M119N 0.2 2.0 2.6 1.8 1.1 0.8 120 D120Y 0.2 1.3 1.7 1.3 1.0 0.7 122 I122R 0.2 2.8

122 I122S 0.2 6.5 6.1 2.6 1.9 0.4 122 I122T 0.3 1.8 1.8 1.1 1.4 0.9 123 N123A 0.8 1.7 1.4 1.1

123 N123C 1.0 1.5 1.4 1.4 0.5 0.1 123 N123G 0.5 1.8 1.6 1.3 0.4 0.1 123 N123S 0.9 1.3 1.2 1.1 0.2 0.1 124 M124A 0.6 1.4 1.0 1.0 1.0 0.7 124 M124H 0.2 1.4 0.3 0.3

124 M124N 0.2 3.0 0.8 0.8

124 M124Q 0.2 5.2 2.6 1.4

124 M124S 0.3 2.8 1.4 1.5 0.9 0.1 124 M124T 0.5 1.8 1.3 1.4 0.9 0.2 124 M124V 1.1 1.4 1.2 1.1 0.9 0.3 125 S125A 0.9 1.9 1.4 1.2 1.1 0.1 126 L126A 0.9 1.6 1.1 1.2

126 L126Q 0.3 3.1 0.9 1.1

126 L126S 0.7 1.6 0.9 0.9

126 L126T 0.5 2.4 1.5 1.4

126 L126Y 0.6 1.5 1.1 1.3

128 G128E 0.3 1.6 1.2 1.5

128 G128N 0.6 1.6 1.6 1.3

128 G128T 0.3 2.2 2.4 1.6

129 P129V 0.7 1.4 1.3 1.1 0.8 0.8 130 S130P 0.6 1.3 1.3 1.0 0.7 0.1 132 S132I 0.3 1.6 1.8 1.3 0.8 0.6 132 S132N 0.9 1.4 1.4 1.2 1.0 0.9 132 S132P 0.2 9.4 11.7  2.5 1.0 0.2 132 S132Q 0.3 1.4 1.3 1.2 0.7 0.9 132 S132V 0.2 3.3 2.4 2.1 0.8 0.6 134 A134I 0.3 1.6 1.2 1.3 0.9 0.9 134 A134L 0.2 1.9 2.4 1.8 1.1 0.6 134 A134M 0.2 1.4 1.2 1.3 1.1 0.5 135 L135I 0.3 1.4 1.0 1.1 0.7 0.2 135 L135T 0.2 3.4 2.3 2.1 0.6 0.1 135 L135V 0.3 2.1 1.1 1.3 0.6 0.2 135 L135W 0.3 2.1 2.0 1.5 0.8 0.2 135 L135Y 0.3 1.9 2.0 1.7

136 K136D 0.2 1.6 1.5 0.9 0.4 0.5 136 K136F 0.3 1.4 1.6 1.2 0.6 0.8 136 K136I 0.2 2.2 2.5 1.4 0.4 0.5 136 K136V 0.2 2.2 2.4 2.0 0.5 0.8 136 K136Y 0.3 1.8 1.4 1.2 0.4 0.9 137 A137P 0.2 3.9 3.4 1.8 0.2 0.1 138 A138D 0.2 1.5 4.6 2.3 0.3 0.2 138 A138E 0.4 1.4 1.0 1.1 0.9 0.8 138 A138F 0.2 2.6 2.7 2.0 0.8 0.3 138 A138H 0.2 2.0 2.2 1.8 0.7 0.5 138 A138Q 0.2 11.9 16.2  5.0 0.9 0.8 138 A138Y 0.2 6.0 9.1 3.3 0.6 0.5 142 A142G 0.5 1.3 1.2 1.0 1.0 1.2 142 A142I 0.3 1.6 1.4 1.5 0.6 0.8 142 A142T 0.3 1.3 1.6 1.5 0.8 1.1 142 A142V 0.4 1.5 1.2 1.2 0.8 1.1 143 V143W 0.7 1.4 0.8 1.0 0.9 0.9 144 A144P 0.2 1.8 2.6 1.2 0.4 0.3 146 G146L 0.2 2.2 3.8 1.9 1.1 0.6 146 G146T 0.3 1.5 1.8 1.6 0.7 1.1 146 G146Y 0.2 1.7 2.2 1.6 0.9 0.9 147 V147D 0.2 2.3 3.1 2.1 1.2 0.5 147 V147P 0.3 1.7 2.1 1.5 0.8 1.1 147 V147W 0.3 2.0 2.2 1.3 0.9 0.9 147 V147Y 0.3 2.3 2.3 1.5 0.9 0.8 148 V148N 0.4 1.4 1.4 1.1 0.8 1.1 148 V148Y 0.2 1.6 1.5 1.6 0.6 0.4 149 V149E 0.2 2.7 3.7 2.5 1.0 0.4 151 A151T 0.5 1.4 1.2 1.2 0.6 0.6 153 A153T 0.2 4.4 4.3 3.0

0.3 153 A153V 0.3 1.3 1.2 1.4 0.3 0.9 159 S159K 1.2 1.4 0.9 1.0 1.0 1.1 164 T164W 0.2 1.6 2.3 1.7 0.1 0.2 165 V165T 0.2 1.5 2.0 1.9 0.1 0.2 167 Y167A 0.7 1.4 1.1 1.3 0.7 0.6 167 Y167D 0.3 1.4 1.6 1.3 0.1 0.1 167 Y167E 0.4 1.5 1.7 1.3 0.5 0.1 167 Y167M 0.2 1.7 1.3 1.4 0.4 0.2 167 Y167P 0.3 1.8 1.9 1.6

167 Y167S 0.3 1.4 1.3 1.3 0.2 0.3 167 Y167T 0.3 1.5 1.3 1.4 0.3 0.4 168 P168L 0.2 2.0 0.9 0.3

168 P168T 0.2 3.0 0.3

169 G169C 0.2 3.0 2.2 2.4 1.0 0.5 170 K170E 0.2 2.9 0.1 1.2

170 K170N 0.3 1.6 1.4 1.2 0.5 0.6 170 K170P 0.2 2.5 2.6 1.6 0.9 0.7 170 K170Q 0.5 1.3 1.2 1.1 0.8 0.6 170 K170S 0.5 1.3 1.1 0.9 0.9 0.8 170 K170T 0.3 1.5 1.5 1.1 0.5 0.5 170 K170Y 0.4 1.4 1.2 1.0 0.9 0.7 171 Y171C 0.2 12.3 8.6 1.8 0.8 0.1 171 Y171D 0.2 4.6 3.5 1.1

171 Y171L 0.2 4.1 3.4 2.4 0.3 0.3 171 Y171N 0.2 2.2

0.5

171 Y171W 0.5 1.4 1.0 1.0 0.6 0.4 172 P172E 0.8 1.3 1.2 1.0 1.2 1.0 172 P172G 0.3 1.4 1.2 1.2 1.0 0.9 172 P172I 0.5 1.4 1.1 1.1 0.9 1.1 172 P172L 0.5 1.5 1.1 1.1 1.0 1.0 172 P172V 0.5 1.4 1.2 1.2 0.8 1.2 172 P172Y 0.3 1.7 1.3 1.4 0.8 0.9 173 S173H 0.5 1.4 0.9 1.2 0.8 0.9 173 S173W 0.3 1.9 1.3 1.5 0.6 0.9 173 S173Y 0.4 1.7 1.0 1.4 0.7 0.9 174 V174A 0.5 1.4 1.2 1.1 0.8 1.0 174 V174I 0.4 1.5 0.9 1.1 0.6 0.6 174 V174L 0.2 93.0 66.8  4.2 0.5 0.3 174 V174S 0.4 1.4 1.0 1.2 0.9 0.8 175 I175A 0.2 6.1 9.3 1.8 0.4 0.3 175 I175F 0.3 1.7 1.2 1.3 0.6 0.8 175 I175R 0.2 1.5

0.1

175 I175T 0.3 2.0 1.6 1.4 0.5 0.9 175 I175V 0.3 2.0 1.3 1.4 0.9 1.1 176 A176V 0.2 1.5

0.6

177 V177W 0.2 3.8 5.4 4.5 0.2 0.5 180 V180F 0.2 1.7

1.5

180 V180H 0.2 2.3 2.9 1.9 0.1 0.1 180 V180Q 0.2 4.6 4.6 2.1

0.3 182 S182W 0.3 1.5 1.4 1.4 0.3 1.0 184 N184A 0.3 1.4 1.9 1.4 0.7 0.7 184 N184L 1.0 1.4 0.9 1.1 0.7 0.9 184 N184V 0.7 1.3 1.1 1.0 0.3 1.0 185 Q185D 0.3 2.0 1.7 1.4 0.6 0.6 185 Q185E 0.8 1.4 1.1 0.9 1.2 1.0 185 Q185I 0.9 1.5 1.0 1.0 0.9 1.2 185 Q185S 0.8 1.4 1.0 1.1 0.8 1.1 185 Q185T 0.8 1.4 1.3 1.1 1.0 1.2 186 R186C 0.4 1.9 1.6 1.1 0.6 0.5 186 R186D 0.2 11.1 16.4  0.2

186 R186E 0.2 23.1 23.9  1.8 0.1 0.1 186 R186F 0.3 1.3 1.4 0.9 0.2 0.3 186 R186G 0.3 2.2 1.9 1.2 0.2 0.6 186 R186I 0.4 1.3 2.1 1.3 0.9 0.8 186 R186L 0.5 1.6 1.4 1.2 0.9 0.9 186 R186N 0.3 2.5 3.7 1.5 0.3 0.4 186 R186P 0.2 3.0 2.3 1.0 0.1 0.2 186 R186Q 0.4 1.5 2.1 1.3 0.8 0.9 186 R186S 0.3 2.4 1.9 1.4 0.3 0.4 186 R186T 0.2 8.6 7.0 2.7 0.4 0.4 186 R186V 0.4 2.0 2.0 1.4 0.9 0.8 186 R186W 0.6 1.5 1.7 1.1 1.1 1.0 186 R186Y 0.4 1.5 2.0 1.4 0.8 0.7 187 A187D 0.9 1.5 1.0 1.0 0.4 0.2 187 A187E 0.7 1.4 1.5 1.0 0.2 0.3 187 A187Q 0.5 1.3 1.2 0.9

0.4 190 S190G 0.3 1.5 2.2 1.3 0.1 0.6 190 S190N 0.2 1.6 2.0 1.1 0.1 0.1 192 Y192D 0.2 1.9 2.2 1.2 0.1 0.1 192 Y192E 0.4 1.4 1.6 1.1

0.2 192 Y192L 0.2 1.9 1.9 1.5

0.2 192 Y192Q 0.4 2.1 1.3 1.2 0.2 0.6 193 G193H 0.2 2.7

193 G193Q 0.2 2.4 0.1 0.1

193 G193T 0.2 4.8 0.5 0.1

193 G193V 0.2 2.3 1.2

194 P194E 0.7 1.5 1.1 1.0 1.0 1.3 194 P194I 0.5 1.6 1.1 1.2 0.9 1.1 194 P194L 0.6 1.6 0.9 1.3 0.9 1.1 194 P194M 0.6 1.4 1.1 1.1 0.8 1.5 194 P194N 0.5 1.8 1.0 1.2 0.1 1.7 194 P194T 0.6 1.3 1.4 1.1 0.9 1.2 194 P194V 0.5 1.7 1.1 1.2 0.8 1.4 195 E195C 0.3 1.4 0.9 0.6 0.4 0.2 195 E195K 0.2 3.7 2.6 2.2 0.3 0.5 195 E195W 0.2 4.3 1.3 1.3 0.1 0.1 196 L196I 0.4 1.7 1.2 1.2 0.8 1.0 196 L196M 0.4 1.5 1.0 1.1 0.2 0.7 196 L196T 0.2 3.9 2.9 2.7 1.0 0.7 196 L196V 0.3 2.0 1.3 1.5 0.7 0.8 197 D197I 0.2 3.1 4.1 2.6

0.3 197 D197M 0.2 1.5 0.7 1.1 0.1 0.1 199 M199F 0.2 28.7 28.8  2.6 0.1 0.2 199 M199Q 0.2 2.1 1.7 1.7 0.8 0.6 200 A200C 0.7 1.3 0.9 1.2 0.6 1.2 200 A200H 0.2 5.0

0.2

200 A200N 0.5 1.3 1.2 1.2 0.7 1.3 200 A200T 0.4 1.7 1.0 1.4 0.2 0.7 200 A200V 0.2 5.1 2.3 2.5

0.4 200 A200Y 0.2 2.6

0.1

201 P201L 0.3 1.7 1.7 2.1

0.6 201 P201T 0.2 4.0 4.1 3.5

0.9 201 P201V 0.2 8.3 11.8  6.0 0.1 0.7 203 V203G 0.2 8.7 7.7 4.9 0.1 0.3 205 I205L 0.3 1.4 1.0 0.9

0.5 205 I205T 0.8 1.6 1.2 1.1 0.8 1.3 208 T208C 0.8 1.5 1.1 1.2 0.8 1.1 208 T208L 0.9 1.4 1.1 1.1

1.2 208 T208M 0.2 5.7 6.0 1.8 0.4 0.4 208 T208P 0.4 1.7 1.1 1.2

0.7 208 T208V 0.6 1.3 1.4 1.2

0.9 209 L209C 0.8 1.4 1.0 1.1 0.9 1.5 209 L209W 0.8 1.4 1.0 1.1 0.3 1.3 210 P210C 0.4 1.5 0.9 1.0 0.2 0.8 210 P210D 0.3 1.4 0.8 1.2 0.1 0.3 210 P210E 0.4 1.7 1.1 1.3 0.4 0.7 210 P210F 0.2 10.3 3.5 2.0 0.1 0.1 210 P210G 0.3 1.8 1.0 1.3

0.6 210 P210Q 0.3 3.0 2.3 2.0

0.8 210 P210R 0.2 3.3 2.8 2.4

0.5 210 P210S 0.6 1.4 1.1 1.2 0.4 1.3 210 P210V 0.5 1.4 1.2 1.1 0.2 0.8 211 G211A 0.9 1.5 1.1 1.0 1.0 0.9 211 G211D 1.0 1.5 1.2 1.0 1.4 1.0 211 G211E 0.9 1.3 1.1 1.1 1.3 0.9 211 G211P 0.9 1.3 1.7 1.0 0.6 1.0 211 G211T 0.8 1.4 1.4 1.2 0.7 1.1 211 G211V 0.8 1.3 1.2 0.9 0.5 0.9 211 G211W 0.8 1.3 1.1 1.2 0.2 1.0 212 N212E 1.0 1.4 1.1 0.9 1.2 0.8 212 N212T 0.2 6.1 4.4 3.0 0.6 1.3 213 K213Q 1.0 1.4 1.0 1.0 1.5 1.0 213 K213T 0.9 1.4 1.2 0.9 1.5 0.9 214 Y214A 0.2 2.4 1.6 0.7 0.2 0.1 214 Y214D 0.2 3.2 3.2 1.7 0.2 0.1 214 Y214N 0.2 1.7 1.6 1.3 0.7 0.2 214 Y214P 0.2 2.1 2.3 1.4

0.7 214 Y214S 0.2 2.2 1.1 1.5 0.2 0.1 215 G215D 0.9 1.3 1.1 1.1 1.0 0.7 215 G215Q 0.8 1.4 1.0 1.0 0.1 0.7 215 G215V 0.4 1.5 0.9 1.2 0.1 0.5 216 A216E 1.2 1.4 0.9 1.0 1.4 0.8 217 Y217E 1.0 1.4 1.0 1.2 1.3 0.5 217 Y217L 1.0 1.3 1.0 1.1 1.4 3.5 217 Y217M 0.9 1.3 1.2 1.1 1.5 1.8 218 N218P 0.3 1.6 0.8 1.2 0.2 0.6 223 A223W 0.2 1.5 0.8 0.2

224 S224D 0.2 1.6 0.6 0.3

224 S224N 0.4 1.8 1.6 1.3 0.6 0.2 224 S224Q 0.3 2.0 0.1 0.2

226 H226E 0.2 1.5 1.9 1.2

0.5 226 H226T 0.4 1.4 1.4 1.2

0.9 227 V227G 0.3 1.6 1.4 1.1 0.9 0.7 227 V227L 0.5 1.4 1.3 1.0 0.4 0.9 227 V227S 0.2 14.7 19.5  4.2 0.5 0.5 230 A230H 0.2 10.4 16.2  3.8 0.1 0.4 230 A230N 0.2 1.8 2.2 1.7 0.6 0.6 231 A231W 0.2 4.4 5.9 4.4 0.3 0.2 231 A231Y 0.3 1.5 2.1 1.6 0.2 0.4 232 A232N 0.2 1.6 2.2 1.5 1.1 0.4 234 I234W 0.2 4.8 6.8 2.4 0.1 0.2 235 L235N 0.3 1.3 1.5 1.2 1.2 0.7 236 S236W 0.2 2.1 3.4 2.3 0.8 0.5 238 H238A 0.3 1.3 1.4 1.1 1.1 0.5 238 H238G 0.2 5.2 6.0 2.4 1.0 0.4 238 H238I 0.2 7.7 7.0 3.6 1.1 0.5 239 P239H 0.9 1.4 1.0 1.1 1.1 1.1 239 P239S 0.9 1.3 1.0 1.0 1.1 1.3 241 W241G 0.3 1.4 1.1 0.9 1.0 0.6 241 W241Q 0.7 1.4 1.0 1.1 1.1 1.1 247 R247H 0.3 1.3 1.5 1.3 0.3 0.7 247 R247L 0.3 1.7 1.5 1.3 0.1 0.6 247 R247W 0.3 1.4 1.4 1.0 0.3 0.7 247 R247Y 0.3 1.5 2.2 1.5 0.2 0.9 250 L250E 0.2 2.6

0.1

250 L250T 0.3 1.5 2.3 1.7 0.8 0.8 252 N252Q 1.4 1.3 1.1 1.1 1.1 1.0 253 T253Y 0.2 3.5 2.0 1.6 0.2 0.1 254 T254D 0.3 2.3 1.8 1.7

0.5 254 T254Q 0.2 32.6 41.1  3.7

0.5 254 T254R 0.2 7.7 3.7 1.6

0.6 255 T255L 1.0 1.5 1.0 1.0

1.0 255 T255P 0.2 1.6 1.0 1.4

0.4 256 K256G 0.7 1.3 0.9 1.0

1.0 256 K256R 1.1 1.4 1.0 1.1

0.9 258 G258P 0.3 1.4 1.6 1.6

0.7 263 Y263D 0.3 1.6 1.4 1.2

0.7 263 Y263K 0.2 2.4 2.8 1.9 0.1 0.3 263 Y263R 0.2 8.0 4.1 1.7 0.1 0.3 265 K265P 0.3 1.6 1.9 1.6 1.0 0.8 268 I268S 0.3 1.4 1.3 1.6

0.8 268 I268T 0.3 1.9 2.0 1.8 0.8 0.4 268 I268W 0.2 2.8 2.4 0.2

270 V270F 0.2 2.4 2.9 1.1 0.2 0.1 273 A273K 0.2 1.6 1.6 1.9

0.6 273 A273P 0.3 1.5 1.7 1.8

0.7 273 A273R 0.2 1.4 1.2 2.0

0.6 273 A273V 0.5 1.4 1.2 1.2 0.3 0.8 273 A273W 0.3 2.1 2.0 1.5

0.8 274 A274W 0.4 1.3 0.7 1.1 0.5 0.7

Table 31-4

-   Parent: FNA (SEQ ID NO:2 with Y217L mutation) -   Measure: PI versus FNA for BMI pH8 16C assay -   Detergent: As indicated in table 31-1 (NA Detergent was Commercial     heat deactivated Tide 2×; WE detergent was commercial heat     deactivated Western European Henkel Persil liquid laundry and Japan     laundry was heat deactivated Japanese Tide laundry powder     detergent).

Generation of Combinatorial Variants of BPN′-Y217L Involving Charged Residue Substitutions

The pAC-FNAre plasmid in FIG. 9 containing the BPN′-Y217L gene was sent to DNA 2.0 Inc. (Menlo Park, Calif.) for the generation of combinatorial variants containing substitutions involving charge changes. They were also provided with the Bacillus subtilis strain (genotype: ΔaprE, ΔnprE, ΔspoIIE, amyE::xylRPxylAcomK-phleo) for transformations. The subtilisin BPN′-Y217L combinatorial charge variants were designed by identifying four well-distributed, surface-exposed, uncharged polar amino-acid residues outside the active site. These residues are Ser-87, Asn-109, Ser-188, and Ser-248. The variants were supplied as glycerol stocks from DNA 2.0. B. subtilis transformants containing BPN′ variant proteins were replicated with a steel 96-well replicator from glycerol stocks into 96-well culture plates (BD, 353075) containing 200 μl of LB media+25 μg/ml chloramphenicol, grown overnight at 37° C., 220 rpm in a humidified enclosure. 200 μl from the overnight culture was used to inoculate 2000 μl MBD defined media+25 μg/ml chloramphenicol in 5 ml plastic shake tubes. MBD (a MOPS based defined medium) was made essentially as known in the art (See, Neidhardt et al., J. Bacteriol., 119: 736-747 [1974]), except that NH₄Cl, FeSO₄, and CaCl₂ were omitted from the base medium, 3 mM K₂HPO₄ was used, and the base medium was supplemented with 60 mM urea, 75 g/L glucose, and 1 soytone. The micronutrients were made up as a 100× stock solution containing in one liter, 400 mg FeSO₄ 7H₂O, 100 mg MnSO₄.H₂O, 100 mg ZnSO₄ 7H₂O, 50 mg CuCl₂ 2H₂O, 100 mg CoCl₂ 6H₂O, 100 mg NaMoO₄ 2H₂O, 100 mg Na₂B₄O₇ 10H₂O, 10 ml of 1M CaCl₂, and 10 ml of 0.5 M sodium citrate. Shake tubes were incubated at 37° C., 220 rpm, for 60 hours. Following 60 hours, culture broth was spun down in a centrifuge at greater than 8000× RCF. The supernatant solution was decanted into 15 ml polypropylene conical tubes for storage. No further purification or concentration was performed. Supernatant stocks were formulated to 40% propylene glycol for long-term stability.

Description

This example describes the testing of BPN′-Y217L variants in BMI microswatch assays in detergents representing various market geographies (e.g., differing pH, T, and/or water hardness), in laundry applications, as described in Test Method 2.

TABLE 31-4 Performance of combinatorial variants of BPN′-Y217L involving charged residue substitutions Net Laundry PI WE Laundry Variants Charge PI NA Laundry PI JPN N109D-Y217L-S248R 0 1.2 1.9 1.6 N109D-S188R-Y217L 0 1.3 1.5 1.3 S87D-Y217L-S248R 0 1.2 1.5 1.5 S87R-N109D-Y217L-S248R 1 1.2 1.5 2.3 S87R-N109D-S188D-Y217L- 0 1.2 1.5 1.0 S248R

Table 31-5: Wash Performance of Selected Double Mutant Proteases

-   Parent: BPN′ wild type (SEQ ID NO: 2) -   Measure: PI at least 1.3 for BMI assay pH8 16C versus wild type (SEQ     ID NO:2) -   Detergent: Commercial heat deactivated Tide 2× Cold

Description

The BPN′ double mutants shown in Table 31-5 below were produced by DNA 2.0 (Menlo Park, Calif.), using BPN′ as the parent gene contained in the expression plasmid pHPLT-BPN′ partial opt. Plasmid pHPLT-BPN′ partial opt was also created by DNA 2.0 (Menlo Park, Calif.) using a codon optimized gene encoding the BPN′ protease and the pHPLT-BPN′ plasmid described above (also see FIG. 3).

The DNA sequence of the pHPLT-BPN′ expression cassette (aprE-BPN′ hybrid leader, BPN′ pro and BPN′ mature DNA sequence from B. amyloliquefaciens) provided below, encodes the BPN′ precursor protein:

GTGAGAAGCAAAAAATTGTGGATCAGTTTGCTGTTTGCTTTAGCGTTAAT CTTTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAAT CAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACG ATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGT GCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAA AAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAA GATCACGTAGCACATGCGTAC GCGCAGTCCGTGCCTTACGGCGTATCACA AATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTA AAGTAGCGGTTATCGACAGCGGTATCGACTCGAGCCATCCAGATCTTAAA GTCGCTGGAGGGGCTTCTATGGTGCCGTCCGAAACAAACCCGTTTCAAGA TAACAATTCTCATGGCACACACGTCGCAGGAACGGTTGCGGCGTTAAACA ATTCTATTGGCGTGCTTGGTGTAGCCCCGTCTGCTTCGCTCTACGCCGTT AAAGTTCTTGGCGCAGACGGATCAGGCCAATACTCATGGATTATCAACGG CATCGAATGGGCCATCGCGAATAACATGGATGTAATCAACATGAGCCTGG GAGGACCAAGCGGCAGTGCGGCACTTAAAGCAGCAGTTGATAAAGCTGTT GCATCTGGTGTCGTCGTAGTAGCGGCAGCTGGGAATGAGGGAACATCCGG ATCATCGAGTACCGTCGGTTATCCAGGCAAGTACCCTTCAGTGATTGCAG TGGGCGCTGTAGACTCTTCAAATCAACGTGCCTCTTTTTCCTCCGTGGGA CCGGAGCTGGATGTCATGGCCCCTGGCGTTTCTATTCAATCGACGCTTCC AGGGAACAAGTATGGTGCGTATAACGGGACTTCCATGGCCTCGCCGCATG TAGCTGGGGCGGCCGCATTGATTCTTTCTAAGCACCCGAACTGGACAAAC ACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTC TTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAG

In the above sequence, bold indicates the DNA that encodes the mature protease, standard font indicates the leader sequence (aprE-BPN′ hybrid leader), and the underlined indicates the pro sequences (BPN′). The amino acid sequence of the mature BPN′ protease is SEQ ID NO:2. The plasmid map

The BPN′ variant proteins were also produced as described earlier. Protein concentration of culture supernatants was determined by TCA precipitation as described in TCA assay of Test Method 2. The stain removal performance of the variants was tested in laundry applications on EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C., pH 7/16° C. and pH 8/32° C. using methods described in Test Method 2 assay methods, with the following modifications. The test detergent used was heat inactivated TIDE® 2× Cold detergent (Proctor & Gamble). Heat inactivation of commercial detergent formulas serves to destroy the endogenous enzymatic activity of any protein components while retaining the properties of nonenzymatic components. Heat inactivation of the detergents was performed by placing pre-weighed amounts of liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The detergent was purchased from local supermarket stores. Both unheated and heated detergents were assayed within 5 minutes of dissolving the detergent, in order to accurately determine percentage deactivated. Functionality of BPN′ variants was quantified as a performance index (PI) (i.e., the ratio of performance of a variant relative to parent BPN′). Results are shown in Table 31-5—in this Table, “ND” indicates “not determined.” Data are shown for variants having a PI of at least 1.3 for BMI pH8 16C cleaning assay.

TABLE 31-5 PI Values of BPN′ Variants Tested for Expression (TCA) and Stain Removal Performance (BMI pH 8/16° C., BMI pH 7/16° C., and BMI pH 8/32° C.) BMI BMI BMI pH 8/ pH pH Variant TCA 16° C. 7/16° C. 8/32° C. G128A-Y217Q 1.2 1.4 1.1 1.2 I111V-M124V 0.9 1.3 ND ND M124V-Y217Q 1.5 1.4 1.1 1.2 N62Q-G97A 1.5 1.3 1.2 1.2 S89Y-M124V 1.1 1.3 ND ND V68A-A92G 0.8 1.4 ND ND V68A-G97A 2.1 1.3 ND ND V68A-I111V 2.1 1.3 ND ND V68A-S89Y 1.7 1.4 ND ND V68A-V227T 1.0 1.4 ND ND V68A-Y217Q 2.3 1.3 ND ND W106F-Y217Q 0.9 1.4 ND ND

Table 31-6: Wash Performance of Selected Series 1 BPN′ Cold Water Proteases

-   Parent: BPN′ wild type (SEQ ID NO: 2) -   Measure: PI at least 1.3 for BMI assay pH8 16C versus wild type (SEQ     ID NO:2) -   Detergent: Commercial heat deactivated Tide 2× Cold

Description

BPN′ multiple mutation libraries (or combinatorial libraries) were produced by Geneart or DNA 2.0, using BPN′ as the parent protein. Protein concentration of culture supernatants was determined by TCA precipitation as described in Test Method 2. The stain removal performance of the variants was tested in laundry applications on EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C., pH 7/16° C. and pH 8/32° C. using methods described in Test Method 2, with the following modifications. The test detergent used was heat inactivated TIDE® 2× Cold detergent (Proctor & Gamble), prepared as described in Test Method 2. Heat inactivation of commercial detergent formulas serves to destroy the endogenous enzymatic activity of any protein components while retaining the properties of non-enzymatic components. Heat inactivation of the detergents was performed by placing pre-weighed amounts of liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The detergent was purchased from local supermarket stores. Both unheated and heated detergents were assayed within 5 minutes of dissolving the detergent, in order to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay. Functionality of BPN′ variants was quantified as a performance index (PI) (i.e., the ratio of performance of a variant relative to parent BPN′). Results are shown in Table 31-6. Performance indices less than or equal to 0.1 were fixed to 0.1 and indicated in bold italics in the table. For every variant with a TCA protein performance index less than or equal to 0.1, all values were fixed at 0.1.

TABLE 31-6 PI Values of BPN′ Variants Tested for Expression (TCA) and Stain Removal Performance (BMI pH 8/16° C., BMI pH 7/16° C., and BMI pH 8/32° C.) BMI BMI BMI pH pH pH Variant TCA 8/16° C. 7/16° C. 8/32° C. G97A-G128A-Y217Q 1.3 1.4 1.2 1.2 G97A-L126A-Y217Q 1.1 1.3 1.2 1.2 G97A-M124V-L126A-Y217Q 1.8 1.4 1.2 1.2 G97A-N123G-Y217Q 0.6 1.4 1.2 1.1 L96T-G97A-Y217Q 0.5 1.3 1.1 1.1 M124V-L126A-Y217Q 1.7 1.4 1.2 1.2 N62Q-G128A-Y217Q 0.7 1.3 1.2 1.1 N62Q-G97A-Y217Q 1.7 1.4 1.2 1.3

Table 31-7: Wash Performance of Selected Series 1 BPN′ Cold Water Protease Variants

-   Parent: FNA (SEQ ID NO:2 with Y217L mutation) -   Measure: PI versus FNA for BMI pH8 16C assay -   Detergent: Commercial heat deactivated Tide 2× Cold

Description

Saturation libraries at positions 97-128-217 in BPN′ (parent) were produced by DNA 2.0. Protein concentration of culture supernatants was determined by TCA precipitation as described in Test Method 2. The stain removal performance of the variants was tested in laundry applications on EMPA 116 swatches (BMI stain, CFT) at pH8/16° C. using methods described in Test Method 2. Functionality of BPN′ variants was quantified as a performance index (PI) (i.e., the ratio of performance of a variant relative to FNA). Results are shown in Table 31-7.

TABLE 31-7 PI Values of BPN′ Variants Tested for Protein Determination (TCA) and Stain Removal Performance (BMI pH 8/16° C.) Variants TCA BMI pH 8 16° C. G97N-G128A-Y217M 1.1 1.4 G97G-G128S-Y217E 1.5 1.4 G97A-G128A-Y217Q 1.3 1.4 G97M-G128S-Y217E 1.2 1.4 G97A-G128S-Y217Q 1.9 1.3 G97D-G128S-Y217Q 1.6 1.3 G97M-G128G-Y217M 1.6 1.3 G97G-G128S-Y217Q 1.6 1.3 G97S-G128S-Y217Q 1.5 1.3 G97G-G128A-Y217Q 1.3 1.3 G97S-G128A-Y217E 1.0 1.3 G97A-G128S-Y217L 2.2 1.3 G97A-G128A-Y217N 1.2 1.3 G97Q-G128S-Y217L 1.9 1.3 G97A-G128A-Y217M 1.5 1.3 G97A-G128A-Y217S 1.4 1.3 G97D-G128A-Y217Q 1.1 1.3 G97M-G128S-Y217Q 1.0 1.3 G97Q-G128G-Y217D-S87Y 1.5 1.3 G97S-G128A-Y217N 1.1 1.3 G97A-G128S-Y217T 1.6 1.3 G97D-G128S-Y217E 1.0 1.3 G97D-G128A-Y217L 1.4 1.3 G97G-G128S-Y217E-S78P-A272T 1.0 1.3 G97T-G128S-Y217D 1.1 1.3 G97D-G128A-Y217I 1.0 1.3 G97Q-G128S-Y217Q 1.6 1.3 G97G-G128A-Y217D 1.1 1.3 G97Q-G128A-Y217N 1.1 1.3 G97S-G128A-Y217M 1.4 1.3 G97S-G128S-Y217N 1.6 1.3 G97S-G128S-Y217M 1.5 1.3 G97E-G128S-Y217M 1.6 1.2 G97S-G128P-Y217Q 1.0 1.2 G97T-G128S-Y217Q 1.1 1.2 G97D-G128S-Y217Q-A73T 1.2 1.2 G97E-G128S-Y217N 1.2 1.2 G97G-G128A-Y217I 1.5 1.2 G97Q-G128A-Y217D 1.1 1.2 G97Q-G128S-Y217M 2.0 1.2 G97R-G128T-Y217Q-S162P 0.7 1.2 G97S-G128S-Y217D 1.5 1.2 G97T-G128P-Y217I 1.3 1.2 G97Q-G128G-Y217E 1.6 1.2 G97C-G128G-Y217N 1.3 1.2 G97D-G128S-Y217H 1.5 1.2 G97M-G128S-Y217L 1.0 1.2 G97M-G128S-Y217N 1.0 1.2 G97S-G128S-Y217E 0.6 1.2 G97M-G128S-Y217I 1.1 1.2 G97A-G128P-Y217A 0.8 1.2 G97R-G128S-Y217D 1.0 1.2

Table 31-8: Wash Performance of Selected Series 1 BPN′ Cold Water Protease Variants

-   Parent: BPN′ v3 (SEQ ID NO:4) -   Measure: PI versus above parent using BMI pH8 16° C. assay -   Detergent: Commercial heat deactivated Tide 2× Cold

Description

Additional BPN′ multiple mutation libraries were produced by Geneart or Gene Oracle, using BPN′: G97A-G128A-Y217Q protein as the parent molecule. Results of experiments conducted to determine stain removal activity (microswatch assay to determine stain removal performance in laundry applications using EMPA 116 swatches (BMI stain, CFT Vlaardingen) (BMI pH8, BMI pH7, BMI 32° C.), protein determination by TCA precipitation, and LAS/EDTA stability (tests of properties of interest) of BPN′ variants are shown in table 31-8. The results were obtained using the methods described in Test Method 2, with the following modifications for the stain removal performance assay. The test detergent used was heat inactivated TIDE® 2× Cold detergent (Proctor & Gamble, Cincinnati, Ohio, USA). Heat inactivation of commercial detergent formulas serves to destroy the endogenous enzymatic activity of any protein components while retaining the properties of non-enzymatic components. Heat inactivation of the detergents was performed by placing pre-weighed amounts of liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The detergent was purchased from local supermarket stores. Both unheated and heated detergents were assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay. As described throughout herein, functionality of BPN′ variants was quantified as a performance index (PI), which is the ratio of performance of a variant to parent protein BPN′: G97A-G128A-Y217Q (i.e. enzyme of SEQ ID NO:4).

TABLE 31-8 Stain removal performance of multiple mutation variants of BPN′ BMI BMI BMI pH 8 pH 7 pH 8 Variant 16 C. 16 C. 32° C. TCA G97A-G128A-Y217Q-S145D 1.1 1.0 1.2 1.0 G97A-G128A-Y217Q-P239R 1.0 1.0 1.1 0.7 G97A-G128A-Y217Q-N61E-P129E- 1.0 0.9 1.1 1.0 S162K-K213L-N240K G97A-G128A-Y217Q-N61E 1.0 1.0 1.1 1.3 G97A-G128A-Y217Q-P40E-A144K- 1.0 0.9 0.9 0.8 K213L G97A-G128A-Y217Q-P129E 1.0 1.0 1.0 1.0 G97A-G128A-Y217Q-N61E-P129E- 1.0 1.0 1.1 1.2 S159K G97A-G128A-Y217Q-K213L 1.0 1.0 1.0 1.1 G97A-G128A-Y217Q-S87D 1.0 1.0 1.0 0.8 G97A-G128A-Y217Q-Q206E 1.0 1.0 1.0 1.0 G97A-G128A-Y217Q-S24R-P40E- 1.0 1.0 1.0 0.9 S145D-S159K-K213L G97A-G128A-Y217Q-K265N 1.0 1.0 1.0 0.9 G97A-G128A-Y217Q-S24R 1.0 0.9 1.0 1.0 G97A-G128A-Y217Q-P40E 1.0 1.0 1.0 0.8 G97A-G128A-Y217Q-Q275E 1.0 1.0 1.0 1.0 G97A-G128A-Y217Q-P129E-S145D- 1.0 1.0 1.0 0.8 N240K G97A-G128A-Y217Q-A144K 1.0 0.9 1.0 0.9 G97A-G128A-Y217Q-S159K 0.9 0.9 1.0 1.0 G97A-G128A-Y217Q-S162K 0.9 0.9 1.0 1.0 G97A-G128A-Y217Q-N240K 0.9 0.9 1.0 0.7

Table 31-9: Wash Performance of Selected Series 1 BPN′ Cold Water Protease Variants

-   Parent: BPN′ v3 (G97A-G128A-Y217Q) -   Measure: PI relative to BPN′ v3 (G97A-G128A-Y217Q) in BMI pH8 16C     assay -   Detergent: Commercial heat deactivated Tide 2× Cold

Description

Additional BPN′ multiple mutation libraries were produced by Geneart or Gene Oracle, using BPN′: G97A-G128A-Y217Q protein as the parent molecule. Results of experiments conducted to determine stain removal activity (microswatch assay to determine stain removal performance in laundry applications using EMPA 116 swatches (BMI stain, CFT Vlaardingen) (BMI pH8, BMI pH7, BMI 32° C.), protein determination by TCA precipitation, and LAS/EDTA stability (tests of properties of interest) of BPN′ variants are shown in table 31-9. The results were obtained using the methods described in Test method 2, with the following modifications for the stain removal performance assay. The test detergent used was heat inactivated TIDE® 2× Cold detergent (Proctor & Gamble, Cincinnati, Ohio, USA). Heat inactivation of commercial detergent formulas serves to destroy the endogenous enzymatic activity of any protein components while retaining the properties of non-enzymatic components. Heat inactivation of the detergents was performed by placing pre-weighed amounts of liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The detergent was purchased from local supermarket stores. Both unheated and heated detergents were assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay. As described throughout herein, functionality of BPN′ variants was quantified as a performance index (PI), which is the ratio of performance of a variant to parent protein BPN′: G97A-G128A-Y217Q (i.e. enzyme of SEQ ID NO:4).

TABLE 31-9 Stain removal performance of multiple mutation variants of BPN′ BMI BMI LAS/ Mutations pH8 pH7 TCA EDTA G97A-G128A-Y217Q-S53G 1.1 1.0 1.2 1.0 G97A-G128A-Y217Q-S78N 1.0 1.0 1.0 1.5 G97A-G128A-Y217Q-S53G-S78N 1.0 1.0 1.1 1.4 G97A-G128A-Y217Q-S53G-I111V 1.0 1.0 0.8 1.2 G97A-G128A-Y217Q-S53G-N117S 0.9 1.0 0.8 1.3 G97A-G128A-Y217Q-S53G-S132N 0.9 0.9 0.7 1.0 G97A-G128A-Y217Q-Y104N-S132N 1.0 1.1 0.7 1.3 G97A-G128A-Y217Q-S53G-S78N-I111V 1.0 1.0 0.9 1.7 G97A-G128A-Y217Q-S53G-S78N-N117S 0.9 0.9 0.7 1.6 G97A-G128A-Y217Q-S53G-S78N-S132N 1.0 0.9 0.8 1.5 G97A-G128A-Y217Q-S53G-Y104N- 0.9 1.0 1.2 1.1 S132N G97A-G128A-Y217Q-S78N-Y104N- 1.0 1.0 0.8 N/D S132N

Table 31-10

-   Parent FNA (BPN′ Y217L) -   Measure PI relative to FNA (BPN′ Y217L) for BMI pH8 16° C. assay     Detergent: Commercial heat-deactivated Tide 2×

Targeted ISD (Insertion Substitution Deletion) Library Construction

A PCR-based method was used to create a library of modified B. amyloliquefaciens subtilisin BPN′-Y217L (commercially available as PURAFECT® PRIME subtilisin) polynucleotides containing in-frame insertions, deletions and/or substitutions in two regions of BPN′-Y217L (positions 63-77 and 92-132 of the mature region) as shown in FIG. 10. This method is basically identical to the described in Pisarchik et al., Prot. Eng. Des. Select., 20:257-265 [2007] with several minor variations. First, we used EamI restriction enzyme to generate sticky ends in our PCR fragments instead of KasI. We also introduced degenerate codon (NDT) right next to the sticky end. Finally, we placed primers much more densely with every following primer being moved only 3 bp downstream until the end of the targeted region was reached. Two sets of oligonucleotides that evenly cover the targeted regions of the BPN′-Y217L gene of a full-length protein of 392 amino acids, in both forward and reverse direction were used to amplify the 5′ and 3′ segments of the portion of the BPN′-Y217L gene. The coding region of the BPN′-Y217L mature protease contains the KpnI and XhoI restriction sites for cloning purposes:

gtgagaagcaaaaaattgtggatca gtttgctgtttgctttagcgt taatctttacgatggcgttcggcagcacatccagcgcgcaggctgcaggg aaatcaaacggggaaaagaaatatattgtcgggtttaaacagacaatgag cacgatgagcgccgctaagaagaaagacgtcatttctgaaaaaggcggga aagtgcaaaagcaattcaaatatgtagacgcagctagcgctacattaaac gaaaaagctgtaaaagaattgaaaaaagacccgagcgtcgcttacgttga agaagatcacgtagcacacgcgtac gcgcagtccgtgccatatggcgtat cacaaattaaagcccctgctctgcactctcaaggctacaccggttcaaat gttaaagtagcggttatcgacagcggtatcgattcttctcatccagatct taaagtagcaggcggagccagcatggttccttctgaaacaaatcctttcc aagacaacaactctcacggaacacacgttgctggtaccgttgcggctctt aataactcaatcggtgtattaggcgttgcgccaagcgcatcactttacgc tgtaaaagttctcggcgccgacggttccggccaatacagctggatcatta acggaatcgagtgggcgatcgcaaacaatatggacgttattaacatgagc ctcggcggaccgtccggttctgctgctttaaaagcggcagttgataaagc cgttgcatccggcgtcgtagtcgttgcggcagccggcaacgaaggcactt ccggcagctcaagcacagtgggctaccctggtaaatacccttctgtcatt gcagtaggcgctgtcgacagcagcaaccaaagagcatctttctcaagcgt aggacctgagctcgatgtcatggcacctggcgtatctatccaaagcacgc ttcctggaaacaaatacggcgcgttgaacggtacatcaatggcatctccg cacgttgccggagccgcggctttgattctttctaagcacccgaactggac aaacactcaagtccgcagctctctagaaaacaccactacaaaacttggtg attctttctactatggaaaagggctgatcaatgtacaggcggcagctcag taa aactcgagataaaaaaccggccttggccccgccggttttttat

The amino acid sequence of the BPN′-Y217L precursor protein is provided below. In this sequence, bold indicates the mature BPN′-Y217L protease:

mrskklwisllfalaliftmafgstssaqaagksngekkyivgfkqtmst msaakkkdvisekggkvqkqfkyvdaasatlnekavkelkkdpsvayvee dhvahayaqsvpygvsqikapalhsqgytgsnvkvavidsgidsshpdlk vaggasmvpsetnpfqdnnshgthvagtvaalnnsigvlgvapsaslyav kvlgadgsgqyswiingiewaiannmdvinmslggpsgsaalkaavdkav asgvvvvaaagnegtsgssstvgypgkypsviavgavdssnqrasfssvg peldvmapgvsiqstlpgnkygalngtsmasphvagaaalilskhpnwtn tqvrsslentttklgdsfyygkglinvqaaaq.

The amino acid sequence of the mature BPN′-Y217L protease was used as the basis for making the variant libraries described herein:

aqsvpygvsqikapalhsqgytgsnvkvavidsgidsshpdlkvaggasm vpsetnpfqdnnshgthvagtvaalnnsigvlgvapsaslyavkvlgadg sgqyswiingiewaiannmdvinmslggpsgsaalkaavdkavasgvvvv aaagnegtsgssstvgypgkypsviavgavdssnqrasfssvgpeldvma pgvsiqstlpgnkygalngtsmasphvagaaalilskhpnwtntqvrssl entttklgdsfyygkglinvqaaaq

Each amplification reaction contained 30 pmol of each oligonucleotide and 100 ng of pAC-FNA10 template DNA (see FIG. 11) Amplifications were carried out using Vent DNA polymerase (New England Biolabs). The PCR mix (20 ul) was initially heated at 95° C. for 2.5 min followed by 30 cycles of denaturation at 94° C. for 15s, annealing at 55° C. for 15s and extension at 72° C. for 40s. Following amplification, left and right fragments generated by the PCR reactions were purified by the Qiagen gel-band purification kit (Valencia, Calif.) and mixed (about 200 ng of each fragment). Every mix contained three left fragments targeting three adjacent codons and three right fragments targeting same codons. These mixes were digested with Eam1104I, ligated with T4 DNA ligase and amplified by flanking primers (P4299 CGTTGAAGAAGATCACGTAGCA, and P3246 TTTATTTTATAAACTCATTCCCTGAT) to generate the full-length gene fragment. The PCR conditions were same as described above, except the extension phase, which was carried out at 72° C. for 2 min The resulting fragments were purified by the Qiagen gel-band purification kit (Valencia, Calif.), digested with MluI and XhoI, and cloned into the MluI/XhoI sites in the Bacillus subtilis expression plasmid pHPLT-FNA.

The BPN′-Y217L expression cassette from the pHPLT vector (Plat promoter-pre-pro-BPN′-Y217L -terminator) used has the polynucleotide sequence shown below.

GCTTTTCTTTTGGAAGAAAATATAGGGAAAATGGTACTTGTTAAAAATTC GGAATATTTATACAATATCATATGTTTCACATTGAAAGGGGAGGAAAATC GTGAAACAACAAAAACGGCTTTAGTCTAGCAAAAGGAGAGGGTAAAGAGT GAGAAGCAAAAAATTGTGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCT TTACGATGGCGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCA AACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCACGAT GAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTGC AAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAAAA GCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAAGA TCACGTAGCACACGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAA TTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAA GTAGCGGTTATCGACAGCGGTATCGATTCTTCTCATCCTGATTTAAAGGT AGCAGGCGGAGCCAGCATGGTTCCTTCTGAAACAAATCCTTTCCAAGACA ACAACTCTCACGGAACTCACGTTGCCGGCACAGTTGCGGCTCTTAATAAC TCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACGCTGTAAA AGTTCTCGGTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAA TCGAGTGGGCGATCGCAAACAATATGGACGTTATTAACATGAGCCTCGGC GGACCTTCTGGTTCTGCTGCTTTAAAAGCGGCAGTTGATAAAGCCGTTGC ATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAGGCACTTCCGGCA GCTCAAGCACAGTGGGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTA GGCGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGCGTAGGACC TGAGCTTGATGTCATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTG GAAACAAATACGGCGCGTTGAACGGTACATCAATGGCATCTCCGCACGTT GCCGGAGCGGCTGCTTTGATTCTTTCTAAGCACCCGAACTGGACAAACAC TCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTCTT TCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTAAACT CGAGAGAGGACGGATTTCCTGAAGGAAATCCGTTTTTTTATTTTAAGCTT G

Ligation mixtures were amplified using rolling circle amplification by Illustra Templiphi kit according to the manufacturer's recommendation (GE Healthcare, Piscataway, N.J.) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, 1 μl of the ligation mixture was mixed with 5 μl of the sample buffer, heated to 95° C. for 3 min and cooled on ice. Next, 5 μl of the reaction buffer and 0.2 μl of the enzyme were added to each tube, followed by incubation at 30° C. for 10 hours. Products of the rolling circle amplification were diluted 100 times and used to transform B. subtilis cells (ΔaprE, ΔnprE, amyE::xylRPxylAcomK-phleo). An aliquot of the transformation mix was plated on LB plates containing 1.6% skim milk and 10 μg/mL neomycin and incubated overnight at 37° C. Subsequently, colonies with halos were inoculated in 120 μl of LB media containing 10 μg/mL neomycin, covered with Enzyscreen lids and incubated overnight at 37° C. and 70% humidity with vigorous shaking (250 rpm). Next morning overnight cultures were frozen and screened later in the BMI assays as described in Test Method 2.

Description

Results of experiments conducted to determine stain removal activity (microswatch assay to determine stain removal performance in laundry applications using EMPA 116 swatches (BMI stain, CFT) at pH 8/16° C. and protein determination by TCA precipitation (tests of properties of interest) of BPN′-Y217L variants are shown in Table 31-10. The results were obtained using the methods described in Test Method 2 with the following modifications for the stain removal performance assay. The test detergent used was heat inactivated Tide 2× detergent (Proctor & Gamble). Heat inactivation of commercial detergent formulas serves to destroy the endogenous enzymatic activity of any protein components while retaining the properties of nonenzymatic components. Heat inactivation of the detergents was performed by placing pre-weighed amounts of liquid detergent (in a glass bottle) in a water bath at 95° C. for 2 hours. The detergent was purchased from local supermarket stores. Both unheated and heated detergents were assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity was tested by AAPF assay. As described throughout functionality of BPN′-Y217L variants was quantified as a performance index (“Pi” or “PI”), which is the ratio of performance of a variant to parent protein BPN′-Y217L.

Data are shown for variants with a PI of at least 1.1 relative to FNA in BMI assays at 16C.

TABLE 31-10 Mutations and Performance Index for TCA protein and BMI Activity for BPN′ Variants PI on BMI at Mutations TCA PI pH 8 16° C. Y217L-V068C-A069G 0.4 1.2 Y217L-I079F-A098G 1.2 1.2 Y217L-P086T-S101D-Q103S-V147I 0.4 1.3 Y217L-A088T-P129S-G146D 0.7 1.2 Y217L-V093I-G128D-P129R 0.6 1.2 Y217L-Z096.01D-A098R 0.7 1.2 Y217L-Z096.01H-A098G 0.3 1.2 Y217L-G097S-Z097.01S-A098G-A273T 0.4 1.2 Y217L-A098S-D099G-G100D 0.3 1.3 Y217L-Z098.01N 0.5 1.3 Y217L-D099G-Z099.01N 0.3 1.2 Y217L-D099G-Z099.01S 0.2 1.2 Y217L-D099V-S101D 0.5 1.2 Y217L-Z099.01S 0.5 1.2 Y217L-G100D 0.5 1.2 Y217L-S101D-Q103H 0.9 1.2 Y217L-S101G-A151V 0.4 1.2 Y217L-S101H-G102S 0.7 1.2 Y217L-S101H-Q103D 1.0 1.2 Y217L-G102R-Q103C-Y104C-V192I 0.3 1.3 Y217L-Q103D 1.0 1.2 Y217L-V121I-I122S-N123C 0.6 1.4 Y217L-V121L-N123C 1.0 1.2 Y217L-I122S-N123S 0.6 1.3 Y217L-M124I 1.7 1.2 Y217L-M124V 1.1 1.2 Y217L-L126F-P129Z-S182N 1.5 1.2 Y217L-L126Y 1.0 1.3 Y217L-G127S-P129D 0.7 1.3 Y217L-Z127.01N-G128S-P129S 0.6 1.2 Y217L-G128H-P129Y 0.4 1.2 Y217L-G128S-P129D 0.9 1.4 Y217L-G128S-P129D-S248R 0.9 1.3 Y217L-G128S-P129G 1.1 1.4 Y217L-P129G-G131Z 0.6 1.2 Y217L-P129G-S130H-S132Z 0.4 1.2 Y217L-P129H-G131Z 0.6 1.2 Y217L-P129L 1.0 1.2 Y217L-P129S-S130H-S132Z 0.5 1.2 Y217L-P129Z 0.6 1.2 Y217L-P129Z-S130G 0.5 1.3 Y217L-P129Z-S130G-G131H-S132H 1.1 1.2 Y217L-P129Z-S130H 0.6 1.2 Y217L-S130V-G131D-S132I 1.0 1.2

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm”.

Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. 

What is claimed is:
 1. A detergent composition comprising A. an isolated protein comprising a protease variant wherein i. the protease variant has at least 95% identity to SEQ ID NO: 2, ii. the protease variant comprises substitutions at positions 24, 53, 78, 101, 128, and 217, and iii. the protease variant has suc-AAPF-pNA proteolytic activity; and B. one or more random graft copolymers.
 2. The detergent composition according to claim 1, wherein said protease variant has a total net charge of −1, 0 or +1 relative to SEQ ID NO:
 2. 3. The detergent composition of claim 1, said composition comprising a material selected from the group consisting of: a surfactant, a builder, a chelating agent, a dye transfer inhibiting agent, a dispersant, one or more additional enzymes, an enzyme stabilizer, a catalytic material, a bleach activator, a hydrogen peroxide, a source of hydrogen peroxide, a preformed peracid, a polymeric dispersing agent, a clay soil removal/anti-redeposition agent, a brightener, a suds suppressor, a dye, a perfume, a perfume delivery system, a structure elasticizing agent, a fabric softener, a carrier, a hydrotrope, a processing aid, a solvent, a pigment and mixtures thereof.
 4. The detergent composition according to claim 1, said composition comprising a second non-immunoequivalent protease that is a subtilisin (EC 3.4.21.62) protease.
 5. The detergent composition of claim 1, said composition comprising an additional enzyme selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, cellobiose dehydrogenases, xyloglucanases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, and mixtures thereof.
 6. The detergent composition of claim 1, said composition comprising a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof said composition being a fabric and home care product. 